siRNA targeting KRAS

ABSTRACT

Efficient sequence specific gene silencing is possible through the use of siRNA technology. By selecting particular siRNAs directed to silencing KRAS, one can maximize the generation of an effective gene silencing reagent, as well as methods for silencing genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

his application is a divisional of U.S. Ser. No. 12,459,670, filed Jul.6, 2009, which is a divisional of U.S. Ser. No. 12/330,981 filed Dec. 9,2008, which is issued as U.S. Pat. No. 7,576,197, and is a divisional ofand claims the benefit of the filing date of U.S. patent applicationSer. No. 11/633,306, filed Dec. 4, 2006 entitled “siRNA Targeting KRAS”which issued as U.S. Pat. No. 7,507,811 and is a continuation of U.S.Ser. No. 10/714,333, filed Nov. 14, 2003, which is a non-provisional ofthe following two provisional Applications: 60/426,137, filed Nov. 14,2002, entitled “Combinatorial Pooling Approach for siRNA Induced GeneSilencing and Method for Selecting siRNA” and U.S. ProvisionalApplication No. 60/502,050, filed Sept. 10, 2003, entitled “Methods forSelecting siRNA.” The disclosures of the aforementioned applications,including the tables submitted in electronic format and sequencelistings are incorporated herein by reference.

SEQUENCE LISTING

The sequence listing for this application has been submitted inaccordance with 37 CFR §1.52(e) and 37 CFR §1.821 on CD-ROM in lieu ofpaper on a disk containing the sequence listing file entitled“DHARMA_(—)0100-US37 CRF.txt” created Apr. 28, 2010,” 89 kb. Applicantshereby incorporate by reference the sequence listing provided on CD-ROMin lieu of paper into the instant specification.

FIELD OF INVENTION

The present invention relates to RNA interference (“RNAi”).

BACKGROUND OF THE INVENTION

Relatively recently, researchers observed that double stranded RNA(“dsRNA”) could be used to inhibit protein expression. This ability tosilence a gene has broad potential for treating human diseases, and manyresearchers and commercial entities are currently investing considerableresources in developing therapies based on this technology.

Double stranded RNA induced gene silencing can occur on at least threedifferent levels: (i) transcription inactivation, which refers to RNAguided DNA or histone methylation; (ii) siRNA induced mRNA degradation;and (iii) mRNA induced transcriptional attenuation.

It is generally considered that the major mechanism of RNA inducedsilencing (RNA interference, or RNAi) in mammalian cells is mRNAdegradation. Initial attempts to use RNAi in mammalian cells focused onthe use of long strands of dsRNA. However, these attempts to induce RNAimet with limited success, due in part to the induction of the interferonresponse, which results in a general, as opposed to a target-specific,inhibition of protein synthesis. Thus, long dsRNA is not a viable optionfor RNAi in mammalian systems.

More recently it has been shown that when short (18-30 bp) RNA duplexesare introduced into mammalian cells in culture, sequence-specificinhibition of target mRNA can be realized without inducing an interferonresponse. Certain of these short dsRNAs, referred to as small inhibitoryRNAs (“siRNAs”), can act catalytically at sub-molar concentrations tocleave greater than 95% of the target mRNA in the cell. A description ofthe mechanisms for siRNA activity, as well as some of its applicationsare described in Provost et al. (2002) Ribonuclease Activity and RNABinding of Recombinant Human Dicer, EMBO J., 21(21): 5864-5874; Tabaraet al. (2002) The dsRNA Binding Protein RDE-4 Interacts with RDE-1,DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell109(7):861-71; Ketting et al., Dicer Functions in RNA Interference andin Synthesis of Small RNA Involved in Developmental Timing in C.elegans; Martinez et al. (2002) Single-Stranded Antisense siRNAs GuideTarget RNA Cleavage in RNAi, Cell 110(5):563; Hutvagner & Zamore (2002)A microRNA in a multiple-turnover RNAi enzyme complex, Science 297:2056.

From a mechanistic perspective, introduction of long double stranded RNAinto plants and invertebrate cells is broken down into siRNA by a TypeIII endonuclease known as Dicer. Sharp (2001) RNA interference—2001,Genes Dev. 15:485. Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs. Bernstein, Caudy, Hammond, & Hannon (2001) Rolefor a bidentate ribonuclease in the initiation step of RNA interference,Nature 409:363. The siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition. Nykanen, Haley, & Zamore (2001) ATP requirements and smallinterfering RNA structure in the RNA interference pathway, Cell 107:309.Upon binding to the appropriate target mRNA, one or more endonucleaseswithin the RISC cleaves the target to induce silencing. Elbashir,Lendeckel, & Tuschl (2001) RNA interference is mediated by 21- and22-nucleotide RNAs, Genes Dev. 15:188, FIG. 1.

The interference effect can be long lasting and may be detectable aftermany cell divisions. Moreover, RNAi exhibits sequence specificity.Kisielow, M. et al. (2002) Isoform-specific knockdown and expression ofadaptor protein ShcA using small interfering RNA, J. of Biochemistry363: 1-5. Thus, the RNAi machinery can specifically-knock down one typeof transcript, while not affecting closely related mRNA. Theseproperties make siRNA a potentially valuable tool for inhibiting geneexpression and studying gene function and drug target validation.Moreover, siRNAs are potentially useful as therapeutic agents against:(1) diseases that are caused by over-expression or misexpression ofgenes; and (2) diseases brought about by expression of genes thatcontain mutations.

Successful siRNA-dependent gene silencing depends on a number offactors. One of the most contentious issues in RNAi is the question ofthe necessity of siRNA design, i.e., considering the sequence of thesiRNA used. Early work in C. elegans and plants circumvented the issueof design by introducing long dsRNA (see, for instance, Fire, A. et al.(1998) Nature 391:806-811). In this primitive organism, long dsRNAmolecules are cleaved into siRNA by Dicer, thus generating a diversepopulation of duplexes that can potentially cover the entire transcript.While some fraction of these molecules are non-functional (i.e. inducelittle or no silencing) one or more have the potential to be highlyfunctional, thereby silencing the gene of interest and alleviating theneed for siRNA design. Unfortunately, due to the interferon response,this same approach is unavailable for mammalian systems. While thiseffect can be circumvented by bypassing the Dicer cleavage step anddirectly introducing siRNA, this tactic carries with it the risk thatthe chosen siRNA sequence may be non-functional or semi-functional.

A number of researches have expressed the view that siRNA design is nota crucial element of RNAi. On the other hand, others in the field havebegun to explore the possibility that RNAi can be made more efficient bypaying attention to the design of the siRNA. Unfortunately, none of thereported methods have provided a satisfactory scheme for reliablyselecting siRNA with acceptable levels of functionality. Accordingly,there is a need to develop rational criteria by which to select siRNAwith an acceptable level of functionality, and to identify siRNA thathave this improved level of functionality, as well as to identify siRNAsthat are hyperfunctional.

SUMMARY OF THE INVENTION

The present invention is directed to increasing the efficiency of RNAi,particularly in mammalian systems. Accordingly, the present inventionprovides kits, siRNAs and methods for increasing siRNA efficacy.

According to one embodiment, the present invention provides a kit forgene silencing, wherein said kit is comprised of a pool of at least twosiRNA duplexes, each of which is comprised of a sequence that iscomplementary to a portion of the sequence of one or more targetmessenger RNA.

According to a second embodiment, the present invention provides amethod for optimizing RNA interference by using one or more siRNAs thatare optimized according to a formula (or algorithm) selected from:

Relative functionality of siRNA=−(GC/3)+(AU ₁₅₋₁₉)−(Tm_(20° C.))*3−(G₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)+(U ₁₀)+(A ₁₄)−(U ₅)−(A ₁₁)   Formula I

Relative functionality of siRNA=−(GC/3)−(AU ₁₅₋₁₉)*3−(G ₁₃)*3−(C ₁₉)+(A₁₉)*2+(A ₃)   Formula II

Relative functionality of siRNA=−(GC/3)+(AU ₁₅₋₁₉)−(Tm_(20° C.))*3  Formula III

Relative functionality of siRNA=−GC/2+(AU ₁₅₋₁₉)/2−(Tm_(20° C.))*2−(G₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)+(U ₁₀)+(A ₁₄)−(U ₅)−(A ₁₁)   Formula IV

Relative functionality of siRNA=−(G ₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)+(U₁₀)+(A ₁₄)−(U ₅)−(A _(n))   Formula V

Relative functionality of siRNA=−(G ₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)  Formula VI

Relative functionality of siRNA=−(GC/2)+(AU ₁₅₋₁₉)/2−(Tm_(20° C.)*)1−(G₁₃)*3−(C ₁₉)+(A ₁₉)*3+(A ₃)*3+(U ₁₀)/2+(A ₁₄)/2−(U ₅)/2−(A ₁₁)/2  Formula VII

wherein in Formulas I-VII:

-   Tm_(20° C.)=1 if the Tm is greater than 20° C.;-   A₁₉=1 if A is the base at position 19 on the sense strand, otherwise    its value is 0;-   AU₁₅₋₁₉=0-5 depending on the number of A or U bases on the sense    strand at positions 15-19;-   G₁₃=1 if G is the base at position 13 on the sense strand, otherwise    its value is 0;-   C₁₉=1 if C is the base at position 19 of the sense strand, otherwise    its value is 0;-   GC=the number of G and C bases in the entire sense strand;-   A₃=1 if A is the base at position 3 on the sense strand, otherwise    its value is 0;-   A₁₁=1 if A is the base at position 11 on the sense strand, otherwise    its value is 0;-   A₁₄=1 if A is the base at position 14 on the sense strand, otherwise    its value is 0;-   U₁₀=1 if U is the base at position 10 on the sense strand, otherwise    its value is 0;-   U₅=1 if U is the base at position 5 on the sense strand, otherwise    its value is 0;

or

Relative functionality of siRNA=(−14)*G ₁₃−13*A ₁−12*U ₇−11*U ₂−10*A₁₁−10*U ₄−10*C _(3-10*) C ₅−10*C ₆−9*A ₁₀−9*U ₉−9*C ₁₈−8*G ₁₀−7*U ₁−7*U₁₆−7*C ₁₇−7*C ₁₉+7*U ₁₇+8*A ₂+8*A ₄+8*A ₅+8*C ₄+9*G ₈+10*A ₇+10*U₁₈+11*A ₁₉+11*C ₉+15*G _(i)+18*A ₃+19*U ₁₀−Tm−3*(GC _(total))−6*(GC₁₅₋₁₉)−30*X; and   Formula VIII

Relative functionality of siRNA=(14.1)*A ₃+(14.9)*A ₆+(17.6)*A₁₃+(24.7)*A ₁₉+(14.2) *U ₁₀+(10.5)*C ₉+(23.9)*G ₁+(16.3)*G ₂+(−12.3)*A₁₁+(−19.3)*U ₁+(−12.1)*U ₂+(−11)*U ₃+(−15.2)*U ₁₅+(−11.3)*U ₁₆+(−11.8)*C₃+(−17.4)*C ₆+(−10.5)*C ₇+(−13.7)*G ₁₃+(−25.9)*G ₁₉−Tm−3*(GC_(total))−6* (GC ₁₅₋₁₉)−30*X

wherein

-   A₁=1 if A is the base at position 1 of the sense strand, otherwise    its value is 0;-   A₂=1 if A is the base at position 2 of the sense strand, otherwise    its value is 0;-   A₃=1 if A is the base at position 3 of the sense strand, otherwise    its value is 0;-   A₄=1 if A is the base at position 4 of the sense strand, otherwise    its value is 0;-   A₅=1 if A is the base at position 5 of the sense strand, otherwise    its value is 0;-   A₆=1 if A is the base at position 6 of the sense strand, otherwise    its value is 0;-   A₇=1 if A is the base at position 7 of the sense strand, otherwise    its value is 0;-   A₁₀=1 if A is the base at position 10 of the sense strand, otherwise    its value is 0;-   A₁₁=1 if A is the base at position 11 of the sense strand, otherwise    its value is 0;-   A₁₃=1 if A is the base at position 13 of the sense strand, otherwise    its value is 0;-   A₁₉=1 if A is the base at position 19 of the sense strand, otherwise    if another base is present or the sense strand is only 18 base pairs    in length, its value is 0;-   C₃=1 if C is the base at position 3 of the sense strand, otherwise    its value is 0;-   C₄=1 if C is the base at position 4 of the sense strand, otherwise    its value is 0;-   C₅=1 if C is the base at position 5 of the sense strand, otherwise    its value is 0;-   C₆=1 if C is the base at position 6 of the sense strand, otherwise    its value is 0;-   C₇=1 if C is the base at position 7 of the sense strand, otherwise    its value is 0;-   C₉=1 if C is the base at position 9 of the sense strand, otherwise    its value is 0;-   C₁₇=1 if C is the base at position 17 of the sense strand, otherwise    its value is 0;-   C₁₈=1 if C is the base at position 18 of the sense strand, otherwise    its value is 0;-   C₁₉=1 if C is the base at position 19 of the sense strand, otherwise    if another base is present or the sense strand is only 18 base pairs    in length, its value is 0;-   G₁=1 if G is the base at position 1 on the sense strand, otherwise    its value is 0;-   G₂=1 if G is the base at position 2 of the sense strand, otherwise    its value is 0;-   G₈=1 if G is the base at position 8 on the sense strand, otherwise    its value is 0;-   G₁₀=1 if G is the base at position 10 on the sense strand, otherwise    its value is 0;-   G₁₃=1 if G is the base at position 13 on the sense strand, otherwise    its value is 0;-   G₁₉=1 if G is the base at position 19 of the sense strand, otherwise    if another base is present or the sense strand is only 18 base pairs    in length, its value is 0;-   U₁=1 if U is the base at position 1 on the sense strand, otherwise    its value is 0;-   U₂=1 if U is the base at position 2 on the sense strand, otherwise    its value is 0;-   U₃=1 if U is the base at position 3 on the sense strand, otherwise    its value is 0;-   U₄=1 if U is the base at position 4 on the sense strand, otherwise    its value is 0;-   U₇=1 if U is the base at position 7 on the sense strand, otherwise    its value is 0;-   U₉=1 if U is the base at position 9 on the sense strand, otherwise    its value is 0;-   U₁₀=1 if U is the base at position 10 on the sense strand, otherwise    its value is 0;-   U₁₅=1 if U is the base at position 15 on the sense strand, otherwise    its value is 0;-   U₁₆=1 if U is the base at position 16 on the sense strand, otherwise    its value is 0;-   U₁₇=1 if U is the base at position 17 on the sense strand, otherwise    its value is 0;-   U₁₈=1 if U is the base at position 18 on the sense strand, otherwise    its value is 0;

GC₁₅₋₁₉=the number of G and C bases within positions 15-19 of the sensestrand or within positions 15-18 if the sense strand is only 18 basepairs in length;

GC_(total)=the number of G and C bases in the sense strand;

Tm=100 if the targeting site contains an inverted repeat longer than 4base pairs, otherwise its value is 0; and

X=the number of times that the same nucleotide repeats four or moretimes in a row.

According to a third embodiment, the present invention is directed to akit comprised of at least one siRNA that contains a sequence that isoptimized according to one of the formulas above. Preferably the kitcontains at least two optimized siRNA, each of which comprises a duplex,wherein one strand of each duplex comprises at least eighteen contiguousbases that are complementary to a region of a target messenger RNA. Formammalian systems, the siRNA preferably comprises between 18 and 30nucleotide base pairs.

The ability to use the above algorithms, which are not sequence orspecies specific, allows for the cost-effective selection of optimizedsiRNAs for specific target sequences. Accordingly, there will be bothgreater efficiency and reliability in the use of siRNA technologies.

According to a fourth embodiment, the present invention provides amethod for developing an siRNA algorithm for selecting functional andhyperfunctional siRNAs for a given sequence. The method comprises:

-   -   (a) selecting a set of siRNAs;    -   (b) measuring the gene silencing ability of each siRNA from said        set;    -   (c) determining the relative functionality of each siRNA;    -   (d) determining the amount of improved functionality by the        presence or absence of at least one variable selected from the        group consisting of the total GC content, melting temperature of        the siRNA, GC content at positions 15-19, the presence or        absence of a particular nucleotide at a particular position and        the number of times that the same nucleotide repeats within a        given sequence; and    -   (e) developing an algorithm using the information of step (d).

According to this embodiment, preferably the set of siRNAs comprises atleast 90 siRNAs from at least one gene, more preferably at least 180siRNAs from at least two different genes, and most preferably at least270 and 360 siRNAs from at least three and four different genes,respectively. Additionally, in step (d) the determination is made withpreferably at least two, more preferably at least three, even morepreferably at least four, and most preferably all of the variables. Theresulting algorithm is not target sequence specific.

In a fifth embodiment, the present invention provides rationallydesigned siRNAs identified using the formulas above.

In a sixth embodiment, the present invention is directed tohyperfunctional siRNA.

For a better understanding of the present invention together with otherand further advantages and embodiments, reference is made to thefollowing description taken in conjunction with the examples, the scopeof which is set forth in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a model for siRNA-RISC interactions. RISC has the abilityto interact with either end of the siRNA or miRNA molecule. Followingbinding, the duplex is unwound, and the relevant target is identified,cleaved, and released.

FIG. 2 is a representation of the functionality of two hundred andseventy siRNA duplexes that were generated to target human cyclophilin,human diazepam-binding inhibitor (DB), and firefly luciferase.

FIG. 3A is a representation of the silencing effect of 30 siRNAs inthree different cells lines, HEK293, DU145, and Hela. FIG. 3B shows thefrequency of different functional groups (>95% silencing (black), >80%silencing (gray), >50% silencing (dark gray), and <50% silencing(white)) based on GC content. In cases where a given bar is absent froma particular GC percentage, no siRNA were identified for that particulargroup. FIG. 3 c shows the frequency of different functional groups basedon melting temperature (Tm). Again, each group has four differentdivisions: >95% (black), >80% (gray), >50% (dark gray), and <50% (white)silencing.

FIG. 4 is a representation of a statistical analysis that revealedcorrelations between silencing and five sequence-related properties ofsiRNA: (A) an A at position 19 of the sense strand, (B) an A at position3 of the sense strand, (C) a U at position 10 of the sense strand, (D) abase other than G at position 13 of the sense strand, and (E) a baseother than C at position 19 of the sense strand. All variables werecorrelated with siRNA silencing of firefly luciferase and humancyclophilin. siRNAs satisfying the criterion are grouped on the left(Selected) while those that do not, are grouped on the right(Eliminated). Y-axis is “% Silencing of Control.” Each position on theX-axis represents a unique siRNA.

FIGS. 5A and 5B are representations of firefly luciferase andcyclophilin siRNA panels sorted according to functionality and predictedvalues using Formula VIII. The siRNA found within the circle representthose that have Formula VIII values (SMARTSCORES™, or siRNA rank) abovezero. SiRNA outside the indicated area have calculated Formula VIIIvalues that are below zero. Y-axis is “Expression (% Control).” Eachposition on the X-axis represents a unique siRNA.

FIG. 6A is a representation of the average internal stability profile(AISP) derived from 270 siRNAs taken from three separate genes(cyclophilin B, DBI and firefly luciferase). Graphs represent AISPvalues of highly functional, functional, and non-functional siRNA. FIG.6B is a comparison between the AISP of naturally derived GFP siRNA(filled squares) and the AISP of siRNA from cyclophilin B, DBI, andluciferase having >90% silencing properties (no fill) for the antisensestrand. “DG” is the symbol for AG, free energy.

FIG. 7 is a histogram showing the differences in duplex functionalityupon introduction of basepair mismatches. The X-axis shows the mismatchintroduced into the siRNA and the position it is introduced (e.g., 8C->Areveals that position 8 (which normally has a C) has been changed to anA). The Y-axis is “% Silencing (Normalized to Control).”

FIG. 8A is histogram that shows the effects of 5′sense and antisensestrand modification with 2′-O-methylation on functionality. FIG. 8B isan expression profile showing a comparison of sense strand off-targeteffects for IGF1R-3 and 2′-O-methyl IGF1R-3. Sense strand off-targets(lower white box) are not induced when the 5′ end of the sense strand ismodified with 2′-O-methyl groups (top white box).

FIG. 9 shows a graph of SMARTSCORES™, or siRNA rank, versus RNAisilencing values for more than 360 siRNA directed against 30 differentgenes. SiRNA to the right of the vertical bar represent those siRNA thathave desirable SMARTSCORES™, or siRNA rank.

FIGS. 10A-E compare the RNAi of five different genes (SEAP, DBI, PLK,Firefly Luciferase, and Renila Luciferase) by varying numbers ofrandomly selected siRNA and four rationally designed (SMART-selected)siRNA chosen using the algorithm described in Formula VIII. In addition,RNAi induced by a pool of the four SMART-selected siRNA is reported attwo different concentrations (100 and 400 nM). 10F is a comparisonbetween a pool of randomly selected EGFR siRNA (Pool 1) and a pool ofSMART selected EGFR siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4represent the individual members that made up each respective pool. Notethat numbers for random siRNAs represent the position of the 5′ end ofthe sense strand of the duplex. The X-axis indicates the duplex that wasapplied. The Y-axis represents the % expression of the control(s).

FIG. 11 shows the Western blot results from cells treated with siRNAdirected against twelve different genes involved in theclathrin-dependent endocytosis pathway (CHC, DynII, CALM, CLCa, CLCb,Eps15, Eps15R, Rab5a, Rab5b, Rab5c, β2 subunit of AP-2 and EEA.1). SiRNAwere selected using Formula VIII. “Pool” represents a mixture ofduplexes 1-4. Total concentration of each siRNA in the pool is 25 nM.Total concentration=4×25=100 nM.

FIG. 12 is a representation of the gene silencing capabilities ofrationally-selected siRNA directed against ten different genes (humanand mouse cyclophilin, C-myc, human lamin A/C, QB (ubiquinol-cytochromec reductase core protein I), MEK1 and MEK2, ATE1 (arginyl-tRNA proteintransferase), GAPDH, and Eg5). The Y-axis is the percent expression ofthe control. Numbers 1, 2, 3 and 4 represent individual rationallyselected siRNA. “Pool” represents a mixture of the four individualsiRNA.

FIG. 13 is the sequence of the top ten Bcl2 siRNAs as determined byFormula VIII. Sequences are listed 5′ to 3′.

FIG. 14 is the knockdown by the top ten Bcl2 siRNAs at 100 nMconcentrations. The Y-axis represents the amount of expression relativeto the non-specific (ns) and transfection mixture control.

FIG. 15 represents a functional walk where siRNA beginning on everyother base pair of a region of the luciferase gene are tested for theability to silence the luciferase gene. The Y-axis represents thepercent expression relative to a control. The X-axis represents theposition of each individual siRNA. Reading from left to right across theX-axis, the position designations are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, and Plasmid.

FIGS. 16A and 16B are histograms demonstrating the inhibition of targetgene expression by pools of 2 (16A) and 3 (16B) siRNA duplexes takenfrom the walk described in FIG. 15. The Y-axis in each represents thepercent expression relative to control. The X-axis in each representsthe position of the first siRNA in paired pools, or trios of siRNAs. Forinstance, the first paired pool contains siRNAs 1 and 3. The secondpaired pool contains siRNAs 3 and 5. Pool 3 (of paired pools) containssiRNAs 5 and 7, and so on. For each of 16A and 16B, the X-axis from leftto right designations are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, and Plasmid.

FIGS. 17A and 17B are histograms demonstrating the inhibition of targetgene expression by pools of 4 (17A) and 5 (17B) siRNA duplexes. TheY-axis in each represents the percent expression relative to control.The X-axis in each represents the position of the first siRNA in eachpool. For each of 17A and 17B, the X-axis from left to right is 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, and Plasmid.

FIGS. 18A and 18B are histograms demonstrating the inhibition of targetgene expression by siRNAs that are ten (18A) and twenty (18B) base pairsapart. The Y-axis represents the percent expression relative to acontrol. The X-axis represents the position of the first siRNA in eachpool. For 18A, the X-axis from left to right designations are 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, and Plasmid. For 18B, the X-axis from leftto right designations are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,and Plasmid.

FIG. 19 shows that pools of siRNAs (dark gray bar) work as well (orbetter) than the best siRNA in the pool (light gray bar). The Y-axisrepresents the percent expression relative to a control. The X-axisrepresents the position of the first siRNA in each pool. The X-axis fromleft to right designations are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, and Plasmid

FIG. 20 shows that the combination of several semifunctional siRNAs(dark gray) result in a significant improvement of gene expressioninhibition over individual (semi-functional; light gray) siRNA. TheX-axis represents the position of the individual siRNAs that weremeasured for effect on expression The Y-axis represents the percentexpression relative to a control.

FIG. 21 show both pools (Library, Lib) and individual siRNAs ininhibition of gene expression of Beta-Galactosidase, Renilla Luciferaseand SEAP (alkaline phosphatase). Numbers on the X-axis indicate theposition of the 5′-most nucleotide of the sense strand of the duplex.The Y-axis represents the percent expression of each gene relative to acontrol. Libraries contain siRNAs that begin at the followingnucleotides: FIG. 21B—Seap: Lib 1: 206, 766, 812, 923, Lib 2: 1117,1280, 1300, 1487, Lib 3: 206, 766, 812, 923, 1117, 1280, 1300, 1487, Lib4: 206, 812, 1117, 1300, Lib 5: 766, 923, 1280, 1487, Lib 6: 206, 1487;FIG. 21A—Bgal: Lib 1 (denoted as I on the figure): 979, 1339, 2029,2590, Lib 2 (denoted as II on the figure): 1087, 1783, 2399, 3257, Lib 3(denoted as III on the figure): 979, 1783, 2590, 3257, Lib 4 (denoted asIV on the figure): 979, 1087, 1339, 1783, 2029, 2399,2590,3257, Lib 5(denoted as Von the figure): 979, 1087, 1339, 1783, Lib 6 (denoted as Ion the figure): 2029, 2399, 2590, 3257; FIG. 21C—Renilla: Lib 1: 174,300, 432, 568, Lib 2: 592, 633, 729, 867, Lib 3: 174, 300, 432, 568,592, 633, 729, 867, Lib 4: 174, 432, 592, 729, Lib 5: 300, 568, 633,867, Lib 6: 592, 568.

FIG. 22 shows the results of an EGFR and TfnR internalization assay whensingle gene knockdowns are performed. The Y-axis represents percentinternalization relative to control.

FIG. 23 shows the results of an EGFR and TfnR internalization assay whenmultiple genes are knocked down (e.g. Rab5a, b, c). The Y-axisrepresents the percent internalization relative to control.

FIG. 24 shows the simultaneous knockdown of four different genes. siRNAsdirected against G6PD, GAPDH, PLK, and UBQ were simultaneouslyintroduced into cells. Twenty-four hours later, cultures were harvestedand assayed for mRNA target levels for each of the four genes. Acomparison is made between cells transfected with individual siRNAs vs.a pool of siRNAs directed against all four genes.

FIG. 25 shows the functionality of ten siRNAs at 0.3nM concentrations.

DETAILED DESCRIPTION Definitions

Unless stated otherwise, the following terms and phrases have themeanings provided below:

Complementary

The term “complementary” refers to the ability of polynucleotides toform base pairs with one another. Base pairs are typically formed byhydrogen bonds between nucleotide units in antiparallel polynucleotidestrands. Complementary polynucleotide strands can base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G), or in any othermanner that allows for the formation of duplexes. As persons skilled inthe art are aware, when using RNA as opposed to DNA, uracil rather thanthymine is the base that is considered to be complementary to adenosine.However, when a U is denoted in the context of the present invention,the ability to substitute a T is implied, unless otherwise stated.

Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can hydrogenbond with a nucleotide unit of a second polynucleotide strand. Less thanperfect complementarity refers to the situation in which some, but notall, nucleotide units of two strands can hydrogen bond with each other.For example, for two 20-mers, if only two base pairs on each strand canhydrogen bond with each other, the polynucleotide strands exhibit 10%complementarity. In the same example, if 18 base pairs on each strandcan hydrogen bond with each other, the polynucleotide strands exhibit90% complementarity.

Deoxynucleotide

The term “deoxynucleotide” refers to a nucleotide or polynucleotidelacking a hydroxyl group (OH group) at the 2′ and/or 3′ position of asugar moiety. Instead, it has a hydrogen bonded to the 2′ and/or 3′carbon. Within an RNA molecule that comprises one or moredeoxynucleotides, “deoxynucleotide” refers to the lack of an OH group atthe 2′ position of the sugar moiety, having instead a hydrogen bondeddirectly to the 2′ carbon.

Deoxlyribonucleotide

The terms “deoxyribonucleotide” and “DNA” refer to a nucleotide orpolynucleotide comprising at least one sugar moiety that has an H,rather than an OH, at its 2′ and/or 3′position.

Duplex Region

The phrase “duplex region” refers to the region in two complementary orsubstantially complementary polynucleotides that form base pairs withone another, either by Watson-Crick base pairing or any other mannerthat allows for a stabilized duplex between polynucleotide strands thatare complementary or substantially complementary. For example, apolynucleotide strand having 21 nucleotide units can base pair withanother polynucleotide of 21 nucleotide units, yet only 19 bases on eachstrand are complementary or substantially complementary, such that the“duplex region” has 19 base pairs. The remaining bases may, for example,exist as 5′ and 3′ overhangs. Further, within the duplex region, 100%complementarity is not required; substantial complementarity isallowable within a duplex region. Substantial complementarity refers to79% or greater complementarity. For example, a mismatch in a duplexregion consisting of 19 base pairs results in 94.7% complementarity,rendering the duplex region substantially complementary.

Gene Silencing

The phrase “gene silencing” refers to a process by which the expressionof a specific gene product is lessened or attenuated. Gene silencing cantake place by a variety of pathways. Unless specified otherwise, as usedherin, gene silencing refers to decreases in gene product expressionthat results from RNA interference (RNAi), a defined, though partiallycharacterized pathway whereby small inhibitory RNA (siRNA) act inconcert with host proteins (e.g. the RNA induced silencing complex,RISC) to degrade messenger RNA (mRNA) in a sequence-dependent fashion.The level of gene silencing can be measured by a variety of means,including, but not limited to, measurement of transcript levels byNorthern Blot Analysis, B-DNA techniques, transcription-sensitivereporter constructs, expression profiling (e.g. DNA chips), and relatedtechnologies. Alternatively, the level of silencing can be measured byassessing the level of the protein encoded by a specific gene. This canbe accomplished by performing a number of studies including WesternAnalysis, measuring the levels of expression of a reporter protein thathas e.g. fluorescent properties (e.g. GFP) or enzymatic activity (e.g.alkaline phosphatases), or several other procedures.

miRNA

The term “miRNA” refers to microRNA.

Nucleotide

The term “nucleotide” refers to a ribonucleotide or adeoxyribonucleotide or modified form thereof, as well as an analogthereof Nucleotides include species that comprise purines, e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs, aswell as pyrimidines, e.g., cytosine, uracil, thymine, and theirderivatives and analogs.

Nucleotide analogs include nucleotides having modifications in thechemical structure of the base, sugar and/or phosphate, including, butnot limited to, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at cytosine exocyclic amines, andsubstitution of 5-bromo-uracil; and 2′-position sugar modifications,including but not limited to, sugar-modified ribonucleotides in whichthe 2′-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH₂,NHR, NR₂, or CN, wherein R is an alkyl moiety. Nucleotide analogs arealso meant to include nucleotides with bases such as inosine, queuosine,xanthine, sugars such as 2′-methyl ribose, non-natural phosphodiesterlinkages such as methylphosphonates, phosphorothioates and peptides.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of types of modifications that cancomprise nucleotides that are modified with respect to the base moietiesinclude but are not limited to, alkylated, halogenated, thiolated,aminated, amidated, or acetylated bases, individually or in combination.More specific examples include, for example, 5-propynyluridine,5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties may be, or be based on,mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose,and other sugars, heterocycles, or carbocycles.

The term nucleotide is also meant to include what are known in the artas universal bases. By way of example, universal bases include but arenot limited to 3-nitropyrrole, 5-nitroindole, or nebularine. The term“nucleotide” is also meant to include the N3′ to P5′ phosphoramidate,resulting from the substitution of a ribosyl 3′ oxygen with an aminegroup.

Further, the term nucleotide also includes those species that have adetectable label, such as for example a radioactive or fluorescentmoiety, or mass label attached to the nucleotide.

Off-Target Silencing and Off-Target Interference

The phrases “off-target silencing” and “off-target interference” aredefined as degradation of mRNA other than the intended target mRNA dueto overlapping and/or partial homology with secondary mRNA messages.

Polynucleotide

The term “polynucleotide” refers to polymers of nucleotides, andincludes but is not limited to DNA, RNA, DNA/RNA hybrids includingpolynucleotide chains of regularly and/or irregularly alternatingdeoxyribosyl moieties and ribosyl moieties (i.e., wherein alternatenucleotide units have an —OH, then and —H, then an —OH, then an —H, andso on at the 2′ position of a sugar moiety), and modifications of thesekinds of polynucleotides, wherein the attachment of various entities ormoieties to the nucleotide units at any position are included.

Polyribonucleotide

The term “polyribonucleotide” refers to a polynucleotide comprising twoor more modified or unmodified ribonucleotides and/or their analogs. Theterm “polyribonucleotide” is used interchangeably with the term“oligoribonucleotide.”

Ribonucleotide and Ribonucleic Acid

The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), referto a modified or unmodified nucleotide or polynucleotide comprising atleast one ribonucleotide unit. A ribonucleotide unit comprises anhydroxyl group attached to the 2′ position of a ribosyl moiety that hasa nitrogenous base attached in N-glycosidic linkage at the 1′ positionof a ribosyl moiety, and a moiety that either allows for linkage toanother nucleotide or precludes linkage.

siRNA

The term “siRNA” refers to small inhibitory RNA duplexes that induce theRNA interference (RNAi) pathway. These molecules can vary in length(generally between 18-30 basepairS) and contain varying degrees ofcomplementarity to their target mRNA in the antisense strand. Some, butnot all, siRNA have unpaired overhanging bases on the 5′ or 3′ end ofthe sense strand and/or the antisense strand. The term “siRNA” includesduplexes of two separate strands, as well as single strands that canform hairpin structures comprising a duplex region.

siRNA may be divided into five (5) groups (non-functional,semi-functional, functional, highly functional, and hyper-functional)based on the level or degree of silencing that they induce in culturedcell lines. As used herein, these definitions are based on a set ofconditions where the siRNA is transfected into said cell line at aconcentration of 100nM and the level of silencing is tested at a time ofroughly 24 hours after transfection, and not exceeding 72 hours aftertransfection. In this context, “non-functional siRNA” are defined asthose siRNA that induce less than 50% (<50%) target silencing.“Semi-functional siRNA” induce 50-79% target silencing. “FunctionalsiRNA” are molecules that induce 80-95% gene silencing.“Highly-functional siRNA” are molecules that induce greater than 95%gene silencing. “Hyperfunctional siRNA” are a special class ofmolecules. For purposes of this document, hyperfunctional siRNA aredefined as those molecules that: (1) induce greater than 95% silencingof a specific target when they are transfected at subnanomolarconcentrations (i.e., less than one nanomolar); and/or (2) inducefunctional (or better) levels of silencing for greater than 96 hours.These relative functionalities (though not intended to be absolutes) maybe used to compare siRNAs to a particular target for applications suchas functional genomics, target identification and therapeutics.

Smartscore™, or siRNA Rank

The term “SMARTSCORE™”, or siRNA rank, refers to a number determined byapplying any of the Formulas I-Formula IX to a given siRNA sequence. Theterm “SMART-selected” or “rationally selected” or “rational selection”refers to siRNA that have been selected on the basis of theirSMARTSCORES™, or siRNA ranking.

Substantially Similar

The phrase “substantially similar” refers to a similarity of at least90% with respect to the identity of the bases of the sequence.

Target

The term “target” is used in a variety of different forms throughoutthis document and is defined by the context in which it is used. “TargetmRNA” refers to a messenger RNA to which a given siRNA can be directedagainst. “Target sequence” and “target site” refer to a sequence withinthe mRNA to which the sense strand of an siRNA shows varying degrees ofhomology and the antisense strand exhibits varying degrees ofcomplementarity. The term “siRNA target” can refer to the gene, mRNA, orprotein against which an siRNA is directed. Similarly “target silencing”can refer to the state of a gene, or the corresponding mRNA or protein.

Transfection

The term “transfection” refers to a process by which agents areintroduced into a cell. The list of agents that can be transfected islarge and includes, but is not limited to, siRNA, sense and/oranti-sense sequences, DNA encoding one or more genes and organized intoan expression plasmid, proteins, protein fragments, and more. There aremultiple methods for transfecting agents into a cell including, but notlimited to, electroporation, calcium phosphate-based transfections,DEAE-dextran-based transfections, lipid-based transfections, molecularconjugate-based transfections (e.g. polylysine-DNA conjugates),microinjection and others.

The present invention is directed to improving the efficiency of genesilencing by siRNA. Through the inclusion of multiple siRNA sequencesthat are targeted to a particular gene and/or selecting an siRNAsequence based on certain defined criteria, improved efficiency may beachieved.

The present invention will now be described in connection with preferredembodiments. These embodiments are presented in order to aid in anunderstanding of the present invention and are not intended, and shouldnot be construed, to limit the invention in any way. All alternatives,modifications and equivalents that may become apparent to those ofordinary skill upon reading this disclosure are included within thespirit and scope of the present invention.

Furthermore, this disclosure is not a primer on RNA interference. Basicconcepts known to persons skilled in the art have not been set forth indetail.

Optimizing siRNA

According to one embodiment, the present invention provides a method forimproving the effectiveness of gene silencing for use to silence aparticular gene through the selection of an optimal siRNA. An siRNAselected according to this method may be used individually, or inconjunction with the first embodiment, i.e., with one or more othersiRNAs, each of which may or may not be selected by this criteria inorder to maximize their efficiency.

The degree to which it is possible to select an siRNA for a given mRNAthat maximizes these criteria will depend on the sequence of the mRNAitself. However, the selection criteria will be independent of thetarget sequence. According to this method, an siRNA is selected for agiven gene by using a rational design. That said, rational design can bedescribed in a variety of ways. Rational design is, in simplest terms,the application of a proven set of criteria that enhance the probabilityof identifying a functional or hyperfunctional siRNA. In one method,rationally designed siRNA can be identified by maximizing one or more ofthe following criteria:

(1) A low GC content, preferably between about 30-52%.

(2) At least 2, preferably at least 3 A or U bases at positions 15-19 ofthe siRNA on the sense strand.

(3) An A base at position 19 of the sense strand.

(4) An A base at position 3 of the sense strand.

(5) A U base at position 10 of the sense strand.

(6) An A base at position 14 of the sense strand.

(7) A base other than C at position 19 of the sense strand.

(8) A base other than G at position 13 of the sense strand.

(9) A Tm, which refers to the character of the internal repeat thatresults in inter- or intramolecular structures for one strand of theduplex, that is preferably not stable at greater than 50° C., morepreferably not stable at greater than 37° C., even more preferably notstable at greater than 30° C. and most preferably not stable at greaterthan 20° C.

(10) A base other than U at position 5 of the sense strand.

(11) A base other than A at position 11 of the sense strand.

Criteria 5, 6, 10 and 11 are minor criteria, but are nonethelessdesirable. Accordingly, preferably an siRNA will satisfy as many of theaforementioned criteria as possible, more preferably at least 1-4 and7-9, and most preferably all of the criteria

With respect to the criteria, GC content, as well as a high number of AUin positions 15-19, may be important for easement of the unwinding ofdouble stranded siRNA duplex. Duplex unwinding has been shown to becrucial for siRNA functionality in vivo.

With respect to criterion 9, the internal structure is measured in termsof the melting temperature of the single strand of siRNA, which is thetemperature at which 50% of the molecules will become denatured. Withrespect to criteria 2-8 and 10-11, the positions refer to sequencepositions on the sense strand, which is the strand that is identical tothe mRNA.

In one preferred embodiment, at least criteria 1 and 8 are satisfied. Inanother preferred embodiment, at least criteria 7 and 8 are satisfied.In still another preferred embodiment, at least criteria 1, 8 and 9 aresatisfied.

It should be noted that all of the aforementioned criteria regardingsequence position specifics are with respect to the 5′ end of the sensestrand. Reference is made to the sense strand, because most databasescontain information that describes the information of the mRNA. Becauseaccording to the present invention a chain can be from 18 to 30 bases inlength, and the aforementioned criteria assumes a chain 19 base pairs inlength, it is important to keep the aforementioned criteria applicableto the correct bases.

When there are only 18 bases, the base pair that is not present is thebase pair that is located at the 3′ of the sense strand. When there aretwenty to thirty bases present, then additional bases are added at the5′ end of the sense chain and occupy positions ⁻1 to ⁻11. Accordingly,with respect to SEQ ID NO 001. NNANANNNNUCNAANNNNA and SEQ ID NO 028.GUCNNANANNNNUCNAANNNNA, both would have A at position 3, A at position5, U at position 10, C at position 11, A and position 13, A and position14 and A at position 19. However, SEQ ID NO 028 would also have C atposition −1, U at position −2 and G at position −3.

For a 19 base pair siRNA, an optimal sequence of one of the strands maybe represented below, where N is any base, A, C, G, or U:

SEQ ID NO 1. NNANANNNNUCNAANNNNA SEQ ID NO 2. NNANANNNNUGNAANNNNASEQ ID NO 3. NNANANNNNUUNAANNNNA SEQ ID NO 4. NNANANNNNUCNCANNNNASEQ ID NO 5. NNANANNNNUGNCANNNNA SEQ ID NO 6. NNANANNNNUUNCANNNNASEQ ID NO 7. NNANANNNNUCNUANNNNA SEQ ID NO 8. NNANANNNNUGNUANNNNASEQ ID NO 9. NNANANNNNUUNUANNNNA SEQ ID NO 10. NNANCNNNNUCNAANNNNASEQ ID NO 11. NNANCNNNNUGNAANNNNA SEQ ID NO 12. NNANCNNNNUUNAANNNNASEQ ID NO 13. NNANCNNNNUCNCANNNNA SEQ ID NO 14. NNANCNNNNUGNCANNNNASEQ ID NO 15. NNANCNNNNUUNCANNNNA SEQ ID NO 16. NANCNNNNUCNUANNNNASEQ ID NO 17. NNANCNNNNUGNUANNNNA SEQ ID NO 18. NNANCNNNNUUNUANNNNASEQ ID NO 19. NNANGNNNNUCNAANNNNA SEQ ID NO 20. NNANGNNNNUGNAANNNNASEQ ID NO 21. NNANGNNNNUUNAANNNNA SEQ ID NO 22. NNANGNNNNUCNCANNNNASEQ ID NO 23. NNANGNNNNUGNCANNNNA SEQ ID NO 24. NNANGNNNNUUNCANNNNASEQ ID NO 25. NNANGNNNNUCNUANNNNA SEQ ID NO 26. NNANGNNNNUGNUANNNNASEQ ID NO 27. NNANGNNNNNUNUANNNNA

In one embodiment, the sequence used as an siRNA is selected by choosingthe siRNA that score highest according to one of the following sevenalgorithms that are represented by Formulas I-VII:

Relative functionality of siRNA=−(GC/3)+(AU ₁₅₋₁₉)−(Tm_(20° C.))*3−(G₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)+(U ₁₀)+(A ₁₄)−(U ₅)−(A ₁₁)   Formula I

Relative functionality of siRNA=−(GC/3)−(AU ₁₅₋₁₉)*3−(G ₁₃)*3−(C ₁₉)+(A₁₉)*2+(A ₃)   Formula II

Relative functionality of siRNA=−(GC/3)+(AU ₁₅₋₁₉)−(Tm_(20° C.))*3  Formula III

Relative functionality of siRNA=−GC/2+(AU ₁₅₋₁₉)/2−(Tm_(20° C.))*2−(G₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)+(U ₁₀)+(A ₁₄)−(U ₅)−(A ₁₁)   Formula IV

Relative functionality of siRNA=−(G ₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)+(U₁₀)+(A ₁₄)−(U ₅)−(A _(n))   Formula V

Relative functionality of siRNA=−(G ₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)  Formula VI

Relative functionality of siRNA=−(GC/2)+(AU ₁₅₋₁₉)/2−(Tm_(20° C.)*)1−(G₁₃)*3−(C ₁₉)+(A ₁₉)*3+(A ₃)*3+(U ₁₀)/2+(A ₁₄)/2−(U ₅)/2−(A ₁₁)/2  Formula VII

In Formulas I-VII:

-   wherein A₁₉=1 if A is the base at position 19 on the sense strand,    otherwise its value is 0,-   AU₁₅₋₁₉=0-5 depending on the number of A or U bases on the sense    strand at positions 15-19;-   G₁₃=1 if G is the base at position 13 on the sense strand, otherwise    its value is 0;-   C₁₉=1 if C is the base at position 19 of the sense strand, otherwise    its value is 0;-   GC=the number of G and C bases in the entire sense strand;-   Tm_(20° C.)=1 if the Tm is greater than 20° C.;-   A₃=1 if A is the base at position 3 on the sense strand, otherwise    its value is 0;-   U₁₀=1 if U is the base at position 10 on the sense strand, otherwise    its value is 0;-   A₁₄=1 if A is the base at position 14 on the sense strand, otherwise    its value is 0;-   U₅=1 if U is the base at position 5 on the sense strand, otherwise    its value is 0; and-   A₁₁=1 if A is the base at position 11 of the sense strand, otherwise    its value is 0.

Formulas I-VII provide relative information regarding functionality.When the values for two sequences are compared for a given formula, therelative functionality is ascertained; a higher positive numberindicates a greater functionality. For example, in many applications avalue of 5 or greater is beneficial.

Additionally, in many applications, more than one of these formulaswould provide useful information as to the relative functionality ofpotential siRNA sequences. However, it is beneficial to have more thanone type of formula, because not every formula will be able to help todifferentiate among potential siRNA sequences. For example, inparticularly high GC mRNAs, formulas that take that parameter intoaccount would not be useful and application of formulas that lack GCelements (e.g., formulas V and VI) might provide greater insights intoduplex functionality. Similarly, formula II might by used in situationswhere hairpin structures are not observed in duplexes, and formula IVmight be applicable for sequences that have higher AU content. Thus, onemay consider a particular sequence in light of more than one or even allof these algorithms to obtain the best differentiation among sequences.In some instances, application of a given algorithm may identify anunusually large number of potential siRNA sequences, and in those cases,it may be appropriate to re-analyze that sequence with a secondalgorithm that is, for instance, more stringent. Alternatively, it isconceivable that analysis of a sequence with a given formula yields noacceptable siRNA sequences (i.e. low SMARTSCORES™, or siRNA ranking). Inthis instance, it may be appropriate to re-analyze that sequences with asecond algorithm that is, for instance, less stringent. In still otherinstances, analysis of a single sequence with two separate formulas maygive rise to conflicting results (i.e. one formula generates a set ofsiRNA with high SMARTSCORES™, or siRNA ranking, while the other formulaidentifies a set of siRNA with low SMARTSCORES™, or siRNA ranking). Inthese instances, it may be necessary to determine which weightedfactor(s) (e.g. GC content) are contributing to the discrepancy andassessing the sequence to decide whether these factors should or shouldnot be included. Alternatively, the sequence could be analyzed by athird, fourth, or fifth algorithm to identify a set of rationallydesigned siRNA.

The above-referenced criteria are particularly advantageous when used incombination with pooling techniques as depicted in Table I:

TABLE I FUNCTIONAL PROBABILITY OLIGOS POOLS CRITERIA >95% >80%<70% >95% >80% <70% CURRENT 33.0 50.0 23.0 79.5 97.3 0.3 NEW 50.0 88.58.0 93.8 99.98 0.005 (GC) 28.0 58.9 36.0 72.8 97.1 1.6

The term “current” refers to Tuschl's conventional siRNA parameters(Elbashir, S. M. et al. (2002) “Analysis of gene function in somaticmammalian cells using small interfering RNAs” Methods 26: 199-213).“New” refers to the design parameters described in Formulas I-VII. “GC”refers to criteria that select siRNA solely on the basis of GC content.

As Table I indicates, when more functional siRNA duplexes are chosen,siRNAs that produce <70% silencing drops from 23% to 8% and the numberof siRNA duplexes that produce >80% silencing rises from 50% to 88.5%.Further, of the siRNA duplexes with >80% silencing, a larger portion ofthese siRNAs actually silence >95% of the target expression (the newcriteria increases the portion from 33% to 50%). Using this new criteriain pooled siRNAs, shows that, with pooling, the amount of silencing >95%increases from 79.5% to 93.8% and essentially eliminates any siRNA poolfrom silencing less than 70%.

Table II similarly shows the particularly beneficial results of poolingin combination with the aforementioned criteria. However, Table II,which takes into account each of the aforementioned variables,demonstrates even a greater degree of improvement in functionality.

TABLE II FUNCTIONAL PROBABILITY OLIGOS POOLS NON- NON- FUNCTIONALAVERAGE FUNCTIONAL FUNCTIONAL AVERAGE FUNCTIONAL RANDOM 20 40 50 67 97 3CRITERIA 1 52 99 0.1 97 93 0.0040 CRITERIA 4 89 99 0.1 99 99 0.0000

The terms “functional,” “Average,” and “Non-functional” refer to siRNAthat exhibit >80%, >50%, and <50% functionality, respectively. Criteria1 and 4 refer to specific criteria described above.

The above-described algorithms may be used with or without a computerprogram that allows for the inputting of the sequence of the mRNA andautomatically outputs the optimal siRNA. The computer program may, forexample, be accessible from a local terminal or personal computer, overan internal network or over the Internet.

In addition to the formulas above, more detailed algorithms may be usedfor selecting siRNA. Preferably, at least one RNA duplex of between 18and 30 base pairs is selected such that it is optimized according aformula selected from:

(−14)*G ₁₃−13*A ₁−12*U ₇−11*U ₂−10*A ₁₁−10*U ₄−10*C _(3-10*) C ₅−10*C₆−9*A ₁₀−9*U ₉−9*C ₁₈−8*G ₁₀−7*U ₁−7*U ₁₆−7*C ₁₇−7*C ₁₉+7*U ₁₇+8*A ₂+8*A₄+8*A ₅+8*C ₄+9*G ₈+10*A ₇+10*U ₁₈+11*A ₁₉+11*C ₉+15*G _(i)+18*A ₃+19*U₁₀−Tm−3*(GC _(total))−6*(GC ₁₅₋₁₉)−30*X; and   Formula VIII

(14.1)*A ₃+(14.9)*A ₆+(17.6)*A ₁₃+(24.7)*A ₁₉+(14.2) *U ₁₀+(10.5)*C₉+(23.9)*G ₁+(16.3)*G ₂+(−12.3)*A ₁₁+(−19.3)*U ₁+(−12.1)*U ₂+(−11)*U₃+(−15.2)*U ₁₅+(−11.3)*U ₁₆+(−11.8)*C ₃+(−17.4)*C ₆+(−10.5)*C₇+(−13.7)*G ₁₃+(−25.9)*G ₁₉−Tm−3*(GC _(total))−6* (GC ₁₅₋₁₉)−30*X

wherein

-   A₁=1 if A is the base at position 1 of the sense strand, otherwise    its value is 0;-   A₂=1 if A is the base at position 2 of the sense strand, otherwise    its value is 0;-   A₃=1 if A is the base at position 3 of the sense strand, otherwise    its value is 0;-   A₄=1 if A is the base at position 4 of the sense strand, otherwise    its value is 0;-   A₅=1 if A is the base at position 5 of the sense strand, otherwise    its value is 0;-   A₆=1 if A is the base at position 6 of the sense strand, otherwise    its value is 0;-   A₇=1 if A is the base at position 7 of the sense strand, otherwise    its value is 0;-   A₁₀=1 if A is the base at position 10 of the sense strand, otherwise    its value is 0;-   A₁₁=1 if A is the base at position 11 of the sense strand, otherwise    its value is 0;-   A₁₃=1 if A is the base at position 13 of the sense strand, otherwise    its value is 0;-   A₁₉=1 if A is the base at position 19 of the sense strand, otherwise    if another base is present or the sense strand is only 18 base pairs    in length, its value is 0;

C₃=1 if C is the base at position 3 of the sense strand, otherwise itsvalue is 0;

-   C₄=1 if C is the base at position 4 of the sense strand, otherwise    its value is 0;-   C₅=1 if C is the base at position 5 of the sense strand, otherwise    its value is 0;-   C₆=1 if C is the base at position 6 of the sense strand, otherwise    its value is 0;-   C₇=1 if C is the base at position 7 of the sense strand, otherwise    its value is 0;-   C₉=1 if C is the base at position 9 of the sense strand, otherwise    its value is 0;-   C₁₇=1 if C is the base at position 17 of the sense strand, otherwise    its value is 0;-   C₁₈=1 if C is the base at position 18 of the sense strand, otherwise    its value is 0;-   C₁₉=1 if C is the base at position 19 of the sense strand, otherwise    if another base is present or the sense strand is only 18 base pairs    in length, its value is 0;

G₁=1 if G is the base at position 1 on the sense strand, otherwise itsvalue is 0;

-   G₂=1 if G is the base at position 2 of the sense strand, otherwise    its value is 0;-   G₈=1 if G is the base at position 8 on the sense strand, otherwise    its value is 0;-   G₁₀=1 if G is the base at position 10 on the sense strand, otherwise    its value is 0;-   G₁₃=1 if G is the base at position 13 on the sense strand, otherwise    its value is 0;-   G₁₉=1 if G is the base at position 19 of the sense strand, otherwise    if another base is present or the sense strand is only 18 base pairs    in length, its value is 0;

U₁=1 if U is the base at position 1 on the sense strand, otherwise itsvalue is 0;

-   U₂=1 if U is the base at position 2 on the sense strand, otherwise    its value is 0;-   U₃=1 if U is the base at position 3 on the sense strand, otherwise    its value is 0;-   U₄=1 if U is the base at position 4 on the sense strand, otherwise    its value is 0;-   U₇=1 if U is the base at position 7 on the sense strand, otherwise    its value is 0;-   U₉=1 if U is the base at position 9 on the sense strand, otherwise    its value is 0;-   U₁₀=1 if U is the base at position 10 on the sense strand, otherwise    its value is 0;-   U₁₅=1 if U is the base at position 15 on the sense strand, otherwise    its value is 0;-   U₁₆=1 if U is the base at position 16 on the sense strand, otherwise    its value is 0;-   U₁₇=1 if U is the base at position 17 on the sense strand, otherwise    its value is 0;-   U₁₈=1 if U is the base at position 18 on the sense strand, otherwise    its value is 0;

GC₁₅₋₁₉=the number of G and C bases within positions 15-19 of the sensestrand, or within positions 15-18 if the sense strand is only 18 basepairs in length;

GC_(total)the number of G and C bases in the sense strand;

Tm=100 if the siRNA oligo has the internal repeat longer then 4 basepairs, otherwise its value is 0; and

X=the number of times that the same nucleotide repeats four or moretimes in a row.

The above formulas VIII and IX, as well as formulas I-VII, providemethods for selecting siRNA in order to increase the efficiency of genesilencing. A subset of variables of any of the formulas may be used,though when fewer variables are used, the optimization hierarchy becomesless reliable.

With respect to the variables of the above-referenced formulas, a singleletter of A or C or G or U followed by a subscript refers to a binarycondition. The binary condition is that either the particular base ispresent at that particular position (wherein the value is “1”) or thebase is not present (wherein the value is “0”). Because position 19 isoptional, i.e. there might be only 18 base pairs, when there are only 18base pairs, any base with a subscript of 19 in the formulas above wouldhave a zero value for that parameter. Before or after each variable is anumber followed by *, which indicates that the value of the variable isto be multiplied or weighed by that number.

The numbers preceding the variables A, or G, or C, or U in Formulas VIIIand IX (or after the variables in Formula I-VII) were determined bycomparing the difference in the frequency of individual bases atdifferent positions in functional siRNA and total siRNA. Specifically,the frequency in which a given base was observed at a particularposition in functional groups was compared with the frequency that thatsame base was observed in the total, randomly selected siRNA set. If theabsolute value of the difference between the functional and total valueswas found to be greater than 6%, that parameter was included in theequation. Thus for instance, if the frequency of finding a “G” atposition 13 (G₁₃) is found to be 6% in a given functional group, and thefrequency of G₁₃ in the total population of siRNAs is 20%, thedifference between the two values is 6%-20%=−14%. As the absolute valueis greater than six (6), this factor (−14) is included in the equation.Thus in Formula VIII, in cases where the siRNA under study has a G inposition 13, the accrued value is (−14)*(1)=−14. In contrast, when abase other than G is found at position 13, the accrued value is(−14)*(0)=0.

When developing a means to optimize siRNAs, the inventors observed thata bias toward low internal thermodynamic stability of the duplex at the5′-antisense (AS) end is characteristic of naturally occurring miRNAprecursors. The inventors extended this observation to siRNAs for whichfunctionality had been assessed in tissue culture.

With respect to the parameter GC₁₅₋₁₉, a value of 0-5 will be ascribeddepending on the number of G or C bases at positions 15 to 19. If thereare only 18 base pairs, the value is between 0 and 4.

With respect to the criterion GC_(total) content, a number from 0-30will be ascribed, which correlates to the total number of G and Cnucleotides on the sense strand, excluding overhangs. Without wishing tobe bound by any one theory, it is postulated that the significance ofthe GC content (as well as AU content at positions 15-19, which is aparameter for formulas III-VII) relates to the easement of the unwindingof a double-stranded siRNA duplex. Duplex unwinding is believed to becrucial for siRNA functionality in vivo and overall low internalstability, especially low internal stability of the first unwound basepair is believed to be important to maintain sufficient processivity ofRISC complex-induced duplex unwinding. If the duplex has 19 base pairs,those at positions 15-19 on the sense strand will unwind first if themolecule exhibits a sufficiently low internal stability at thatposition. As persons skilled in the art are aware, RISC is a complex ofapproximately twelve proteins; Dicer is one, but not the only, helicasewithin this complex. Accordingly, although the GC parameters arebelieved to relate to activity with Dicer, they are also important foractivity with other RISC proteins.

The value of the parameter Tm is 0 when there are no internal repeatslonger than (or equal to) four base pairs present in the siRNA duplex;otherwise the value is 1. Thus for example, if the sequence ACGUACGU, orany other four nucleotide (or more) palindrome exists within thestructure, the value will be one (1). Alternatively if the structureACGGACG, or any other 3 nucleotide (or less) palindrome exists, thevalue will be zero (0).

The variable “X” refers to the number of times that the same nucleotideoccurs contiguously in a stretch of four or more units. If there are,for example, four contiguous As in one part of the sequence andelsewhere in the sequence four contiguous Cs, X=2. Further, if there aretwo separate contiguous stretches of four of the same nucleotides oreight or more of the same nucleotides in a row, then X=2. However, Xdoes not increase for five, six or seven contiguous nucleotides.

Again, when applying Formula VIII or Formula IX to a given mRNA, (the“target RNA” or “target molecule”), one may use a computer program toevaluate the criteria for every sequence of 18-30 base pairs or onlysequences of a fixed length, e.g., 19 base pairs. Preferably thecomputer program is designed such that it provides a report ranking ofall of the potential siRNAs between 18 and 30 base pairs, rankedaccording to which sequences generate the highest value. A higher valuerefers to a more efficient siRNA for a particular target gene. Thecomputer program that may be used, may be developed in any computerlanguage that is known to be useful for scoring nucleotide sequences, orit may be developed with the assistance of commercially availableproduct such as Microsoft's product .net. Additionally, rather than runevery sequence through one and/or another formula, one may compare asubset of the sequences, which may be desirable if for example only asubset are available. For instance, it may be desirable to first performa BLAST (Basic Local Alignment Search Tool) search and to identifysequences that have no homology to other targets. Alternatively, it maybe desirable to scan the sequence and to identify regions of moderate GCcontext, then perform relevant calculations using one of theabove-described formulas on these regions. These calculations can bedone manually or with the aid of a computer.

As with Formulas I-VII, either Formula VIII or Formula IX may be usedfor a given mRNA target sequence. However, it is possible that accordingto one or the other formula more than one siRNA will have the samevalue. Accordingly, it is beneficial to have a second formula by whichto differentiate sequences. Formula IX was derived in a similar fashionas Formula VIII, yet used a larger data set and thus yields sequenceswith higher statistical correlations to highly functional duplexes. Thesequence that has the highest value ascribed to it may be referred to asa “first optimized duplex.” The sequence that has the second highestvalue ascribed to it may be referred to as a “second optimized duplex.”Similarly, the sequences that have the third and fourth highest valuesascribed to them may be referred to as a third optimized duplex and afourth optimized duplex, respectively. When more than one sequence hasthe same value, each of them may, for example, be referred to as firstoptimized duplex sequences or co-first optimized duplexes.

siRNA sequences identified using Formula VIII are contained within thecompact disks provided in parent application Ser. No. 10/714,333. Thedata included on the compact disks is described more fully below. Thesequences identified by Formula VIII that are disclosed in the compactsdisks may be used in gene silencing applications.

It should be noted that for Formulas VIII and IX all of theaforementioned criteria are identified as positions on the sense strandwhen oriented in the 5′ to 3′ direction as they are identified inconnection with Formulas I-VII unless otherwise specified.

Formulas I-IX, may be used to select or to evaluate one, or more thanone, siRNA in order to optimize silencing. Preferably, at least twooptimized siRNAs that have been selected according to at least one ofthese formulas are used to silence a gene, more preferably at leastthree and most preferably at least four. The siRNAs may be usedindividually or together in a pool or kit. Further, they may be appliedto a cell simultaneously or separately. Preferably, the at least twosiRNAs are applied simultaneously. Pools are particularly beneficial formany research applications. However, for therapeutics, it may be moredesirable to employ a single hyperfunctional siRNA as describedelsewhere in this application.

When planning to conduct gene silencing, and it is necessary to choosebetween two or more siRNAs, one should do so by comparing the relativevalues when the siRNA are subjected to one of the formulas above. Ingeneral a higher scored siRNA should be used.

Useful applications include, but are not limited to, target validation,gene functional analysis, research and drug discovery, gene therapy andtherapeutics. Methods for using siRNA in these applications are wellknown to persons of skill in the art.

Because the ability of siRNA to function is dependent on the sequence ofthe RNA and not the species into which it is introduced, the presentinvention is applicable across a broad range of species, including butnot limited to all mammalian species, such as humans, dogs, horses,cats, cows, mice, hamsters, chimpanzees and gorillas, as well as otherspecies and organisms such as bacteria, viruses, insects, plants and C.elegans.

The present invention is also applicable for use for silencing a broadrange of genes, including but not limited to the roughly 45,000 genes ofa human genome, and has particular relevance in cases where those genesare associated with diseases such as diabetes, Alzheimer's, cancer, aswell as all genes in the genomes of the aforementioned organisms.

The siRNA selected according to the aforementioned criteria or one ofthe aforementioned algorithms are also, for example, useful in thesimultaneous screening and functional analysis of multiple genes andgene families using high throughput strategies, as well as in directgene suppression or silencing.

Development of the Algorithms

To identify siRNA sequence features that promote functionality and toquantify the importance of certain currently accepted conventionalfactors—such as G/C content and target site accessibility—the inventorssynthesized an siRNA panel consisting of 270 siRNAs targeting threegenes, Human Cyclophilin, Firefly Luciferase, and Human DBI. In allthree cases, siRNAs were directed against specific regions of each gene.For Human Cyclophilin and Firefly Luciferase, ninety siRNAs weredirected against a 199 by segment of each respective mRNA. For DBI, 90siRNAs were directed against a smaller, 109 base pair region of themRNA. The sequences to which the siRNAs were directed are providedbelow.

It should be noted that in certain sequences, “t” is present. This isbecause many databases contain information in this manner. However, thet denotes a uracil residue in mRNA and siRNA. Any algorithm will, unlessotherwise specified, process a t in a sequence as a u.

Human Cylophilin: 193-390, M60857

SEQ ID NO 029: gttccaaaaa cagtggataa ttttgtggcc ttagctacaggagagaaagg atttggctac aaaaacagca aattccatcgtgtaatcaag gacttcatga tccagggcgg agacttcaccaggggagatg gcacaggagg aaagagcatc tacggtgagcgcttccccga tgagaacttc aaactgaagc actacgggcc tggctggg

Firefly Luciferase: 1434-1631, U47298 (PGL3, PROMEGA)

SEQ ID NO 030: tgaacttccc gccgccgttg ttgttttgga gcacggaaagacgatgacgg aaaaagagat cgtggattac gtcgccagtcaagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtttgtggacgaa gtaccgaaag gtcttaccgg aaaactcgacgcaagaaaaa tcagagagat cctcataaag gccaagaagg

DBI, NM_(—)020548 (202-310) (Every Position)

SEQ ID NO 031: acgggcaagg ccaagtggga tgcctggaat gagctgaaagggacttccaa ggaagatgcc atgaaagctt acatcaacaaagtagaagag ctaaagaaaa aatacggg

A list of the siRNAs appears in Table IV (see Examples Section, ExampleI).

The set of duplexes was analyzed to identify correlations between siRNAfunctionality and other biophysical or thermodynamic properties. Whenthe siRNA panel was analyzed in functional and non-functional subgroups,certain nucleotides were much more abundant at certain positions infunctional or non-functional groups. More specifically, the frequency ofeach nucleotide at each position in highly functional siRNA duplexes wascompared with that of nonfunctional duplexes in order to assess thepreference for or against any given nucleotide at every position. Theseanalyses were used to determine important criteria to be included in thesiRNA algorithms (Formulas VIII and IX).

The data set was also analyzed for distinguishing biophysical propertiesof siRNAs in the functional group, such as optimal percent of GCcontent, propensity for internal structures and regional thermodynamicstability. Of the presented criteria, several are involved in duplexrecognition, RISC activation/duplex unwinding, and target cleavagecatalysis.

The original data set that was the source of the statistically derivedcriteria is shown in FIG. 2. Additionally, this figure shows that randomselection yields siRNA duplexes with unpredictable and widely varyingsilencing potencies as measured in tissue culture using HEK293 cells. Inthe figure, duplexes are plotted such that each x-axis tick-markrepresents an individual siRNA, with each subsequent siRNA differing intarget position by two nucleotides for Human Cyclophilin and FireflyLuciferase, and by one nucleotide for Human DBL Furthermore, the y-axisdenotes the level of target expression remaining after transfection ofthe duplex into cells and subsequent silencing of the target.

siRNA identified and optimized in this document work equally well in awide range of cell types. FIG. 3 a shows the evaluation of thirty siRNAstargeting the DBI gene in three cell lines derived from differenttissues. Each DBI siRNA displays very similar functionality in HEK293(ATCC, CRL-1573, human embryonic kidney), HeLa (ATCC, CCL-2, cervicalepithelial adenocarcinoma) and DU145 (HTB-81, prostate) cells asdeterimined by the B-DNA assay. Thus, siRNA functionality is determinedby the primary sequence of the siRNA and not by the intracellularenvironment. Additionally, it should be noted that although the presentinvention provides for a determination of the functionality of siRNA fora given target, the same siRNA may silence more than one gene. Forexample, the complementary sequence of the silencing siRNA may bepresent in more than one gene. Accordingly, in these circumstances, itmay be desirable not to use the siRNA with highest SMARTSCORE™, or siRNAranking. In such circumstances, it may be desirable to use the siRNAwith the next highest SMARTSCORE™, or siRNA ranking.

To determine the relevance of G/C content in siRNA function, the G/Ccontent of each duplex in the panel was calculated and the functionalclasses of siRNAs (<F50, ≧F50, ≧F80, ≧F95 where F refers to the percentgene silencing) were sorted accordingly. The majority of thehighly-functional siRNAs (>F95) fell within the G/C content range of36%-52% (FIG. 3B). Twice as many non-functional (<F50) duplexes fellwithin the high G/C content groups (>57% GC content) compared to the36%-52% group. The group with extremely low GC content (26% or less)contained a higher proportion of non-functional siRNAs and nohighly-functional siRNAs. The G/C content range of 30%-52% was thereforeselected as Criterion I for siRNA functionality, consistent with theobservation that a G/C range 30%-70% promotes efficient RNAi targeting.Application of this criterion alone provided only a marginal increase inthe probability of selecting functional siRNAs from the panel: selectionof F50 and F95 siRNAs was improved by 3.6% and 2.2%, respectively. ThesiRNA panel presented here permitted a more systematic analysis andquantification of the importance of this criterion than that usedpreviously.

A relative measure of local internal stability is the A/U base pair (bp)content; therefore, the frequency of A/U by was determined for each ofthe five terminal positions of the duplex (5′ sense (S)/5′ antisense(AS)) of all siRNAs in the panel. Duplexes were then categorized by thenumber of A/U by in positions 1-5 and 15-19 of the sense strand. Thethermodynamic flexibility of the duplex 5′-end (positions 1-5; S) didnot appear to correlate appreciably with silencing potency, while thatof the 3′-end (positions 15-19; S) correlated with efficient silencing.No duplexes lacking A/U by in positions 15-19 were functional. Thepresence of one A/U by in this region conferred some degree offunctionality, but the presence of three or more A/Us was preferable andtherefore defined as Criterion II. When applied to the test panel, onlya marginal increase in the probability of functional siRNA selection wasachieved: a 1.8% and 2.3% increase for F50 and F95 duplexes,respectively (Table III).

The complementary strands of siRNAs that contain internal repeats orpalindromes may form internal fold-back structures. These hairpin-likestructures exist in equilibrium with the duplexed form effectivelyreducing the concentration of functional duplexes. The propensity toform internal hairpins and their relative stability can be estimated bypredicted melting temperatures. High Tm reflects a tendency to formhairpin structures. Lower Tm values indicate a lesser tendency to formhairpins. When the functional classes of siRNAs were sorted by T_(m)(FIG. 3C), the following trends were identified: duplexes lacking stableinternal repeats were the most potent silencers (no F95 duplex withpredicted hairpin structure T_(m)>60° C.). In contrast, about 60% of theduplexes in the groups having internal hairpins with calculated T_(m)values less than 20° C. were F80. Thus, the stability of internalrepeats is inversely proportional to the silencing effect and definesCriterion III (predicted hairpin structure T_(m)<20° C.).

Sequence-Based Determinants of siRNA Functionality

When the siRNA panel was sorted into functional and non-functionalgroups, the frequency of a specific nucleotide at each position in afunctional siRNA duplex was compared with that of a nonfunctional duplexin order to assess the preference for or against a certain nucleotide.FIG. 4 shows the results of these queries and the subsequent resortingof the data set (from FIG. 2). The data is separated into two sets:those duplexes that meet the criteria, a specific nucleotide in acertain position—grouped on the left (Selected) and those that donot—grouped on the right (Eliminated). The duplexes are further sortedfrom most functional to least functional with the y-axis of FIG. 4 a-erepresenting the % expression i.e. the amount of silencing that iselicited by the duplex (Note: each position on the X-axis represents adifferent duplex). Statistical analysis revealed correlations betweensilencing and several sequence-related properties of siRNAs. FIG. 4 andTable III show quantitative analysis for the following fivesequence-related properties of siRNA: (A) an A at position 19 of thesense strand; (B) an A at position 3 of the sense strand; (C) a U atposition 10 of the sense strand; (D) a base other than G at position 13of the sense strand; and (E) a base other than C at position 19 of thesense strand.

When the siRNAs in the panel were evaluated for the presence of an A atposition 19 of the sense strand, the percentage of non-functionalduplexes decreased from 20% to 11.8%, and the percentage of F95 duplexesincreased from 21.7% to 29.4% (Table III). Thus, the presence of an A inthis position defined Criterion IV.

Another sequence-related property correlated with silencing was thepresence of an A in position 3 of the sense strand (FIG. 4 b). Of thesiRNAs with A3, 34.4% were F95, compared with 21.7% randomly selectedsiRNAs. The presence of a U base in position 10 of the sense strandexhibited an even greater impact (FIG. 4 c). Of the duplexes in thisgroup, 41.7% were F95. These properties became criteria V and VI,respectively.

Two negative sequence-related criteria that were identified also appearon FIG. 4. The absence of a G at position 13 of the sense strand,conferred a marginal increase in selecting functional duplexes (FIG. 4d). Similarly, lack of a C at position 19 of the sense strand alsocorrelated with functionality (FIG. 4 e). Thus, among functionalduplexes, position 19 was most likely occupied by A, and rarely occupiedby C. These rules were defined as criteria VII and VIII, respectively.

Application of each criterion individually provided marginal butstatistically significant increases in the probability of selecting apotent siRNA. Although the results were informative, the inventorssought to maximize potency and therefore consider multiple criteria orparameters. Optimization is particularly important when developingtherapeutics. Interestingly, the probability of selecting a functionalsiRNA based on each thermodynamic criteria was 2%-4% higher than random,but 4%-8% higher for the sequence-related determinates. Presumably,these sequence-related increases reflect the complexity of the RNAimechanism and the multitude of protein-RNA interactions that areinvolved in RNAi-mediated silencing.

TABLE III IMPROVEMENT PERCENT OVER CRITERION FUNCTIONAL RANDOM (%) I.30%-52% G/C Content <F50 16.4 −3.6 ≧F50 83.6 3.6 ≧F80 60.4 4.3 ≧F95 23.92.2 II. At least 3 A/U <F50 18.2 −1.8 bases at positions ≧F50 81.8 1.815-19 of the sense ≧F80 59.7 3.6 strand ≧F95 24.0 2.3 III. Absence ofinternal <F50 16.7 −3.3 repeats, as measured ≧F50 83.3 3.3 by Tm ofsecondary ≧F80 61.1 5.0 structure ≦20° C. ≧F95 24.6 2.9 IV. An A base at<F50 11.8 −8.2 position 19 ≧F50 88.2 8.2 of the sense strand ≧F80 75.018.9 ≧F95 29.4 7.7 V. An A base at <F50 17.2 −2.8 position 3 of ≧F5082.8 2.8 the sense strand ≧F80 62.5 6.4 ≧F95 34.4 12.7 VI. A U base at<F50 13.9 −6.1 position 10 of ≧F50 86.1 6.1 the sense strand ≧F80 69.413.3 ≧F95 41.7 20 VII. A base other than <F50 18.8 −1.2 C at position 19≧F50 81.2 1.2 of the sense strand ≧F80 59.7 3.6 ≧F95 24.2 2.5 VIII. Abase other than <F50 15.2 −4.8 G at position 13 ≧F50 84.8 4.8 of thesense strand ≧F80 61.4 5.3 ≧F95 26.5 4.8The siRNA Selection Algorithm

In an effort to improve selection further, all identified criteria,including but not limited to those listed in Table III were combinedinto the algorithms embodied in Formula VIII and Formula IX. Each siRNAwas then assigned a score (referred to as a SMARTSCORE™, or siRNAranking) according to the values derived from the formulas. Duplexesthat scored higher than 0 or 20, for Formulas VIII and IX, respectively,effectively selected a set of functional siRNAs and excluded allnon-functional siRNAs. Conversely, all duplexes scoring lower than 0 and20 according to formulas VIII and IX, respectively, contained somefunctional siRNAs but included all non-functional siRNAs. A graphicalrepresentation of this selection is shown in FIG. 5.

The methods for obtaining the seven criteria embodied in Table III areillustrative of the results of the process used to develop theinformation for Formulas VIII and IX. Thus similar techniques were usedto establish the other variables and their multipliers. As describedabove, basic statistical methods were use to determine the relativevalues for these multipliers.

To determine the value for “Improvement over Random” the difference inthe frequency of a given attribute (e.g. GC content, base preference) ata particular position is determined between individual functional groups(e.g. <F50) and the total siRNA population studied (e.g. 270 siRNAmolecules selected randomly). Thus, for instance, in Criterion I(30%-52% GC content) members of the <F50 group were observed to have GCcontents between 30-52% in 16.4% of the cases. In contrast, the totalgroup of 270 siRNAs had GC contents in this range, 20% of the time. Thusfor this particular attribute, there is a small negative correlationbetween 30%-52% GC content and this functional group (i.e. 16.4%-20%=-3.6%). Similarly, for Criterion VI, (a “U” at position 10 of the sensestrand), the >F95 group contained a “U” at this position 41.7% of thetime. In contrast, the total group of 270 siRNAs had a “U” at thisposition 21.7% of the time, thus the improvement over random iscalculated to be 20% (or 41.7%-21.7%).

Identifying the Average Internal Stability Profile of Strong siRNA

In order to identify an internal stability profile that ischaracteristic of strong siRNA, 270 different siRNAs derived from thecyclophilin B, the diazepam binding inhibitor (DBI), and the luciferasegene were individually transfected into HEK293 cells and tested fortheir ability to induce RNAi of the respective gene. Based on theirperformance in the in vivo assay, the sequences were then subdividedinto three groups, (i) >95% silencing; (ii) 80-95% silencing; and (iii)less than 50% silencing. Sequences exhibiting 51-84% silencing wereeliminated from further consideration to reduce the difficulties inidentifying relevant thermodynamic patterns.

Following the division of siRNA into three groups, a statisticalanalysis was performed on each member of each group to determine theaverage internal stability profile (AISP) of the siRNA. To accomplishthis the Oligo 5.0 Primer Analysis Software and other relatedstatistical packages (e.g. Excel) were exploited to determine theinternal stability of pentamers using the nearest neighbor methoddescribed by Freier et al., (1986) Improved free-energy parameters forpredictions of RNA duplex stability, Proc Natl. Acad. Sci. USA 83(24):9373-7. Values for each group at each position were then averaged, andthe resulting data were graphed on a linear coordinate system with theY-axis expressing the AG (free energy) values in kcal/mole and theX-axis identifying the position of the base relative to the 5′ end.

The results of the analysis identified multiple key regions in siRNAmolecules that were critical for successful gene silencing. At the3′-most end of the sense strand (5′antisense), highly functional siRNA(>95% gene silencing, see FIG. 6A, >F95) have a low internal stability(AISP of position 19=˜−7.6 kcal/mol). In contrast low-efficiency siRNA(i.e. those exhibiting less than 50% silencing, <F50) display adistinctly different profile, having high AG values (˜−8.4 kcal/mol) forthe same position. Moving in a 5′ (sense strand) direction, the internalstability of highly efficient siRNA rises (position 12=˜−8.3 kcal/mole)and then drops again (position 7=˜−7.7 kcal/mol) before leveling off ata value of approximately −8.1 kcal/mol for the 5′ terminus. siRNA withpoor silencing capabilities show a distinctly different profile. Whilethe AISP value at position 12 is nearly identical with that of strongsiRNAs, the values at positions 7 and 8 rise considerably, peaking at ahigh of ˜−9.0 kcal/mol. In addition, at the 5′ end of the molecule theAISP profile of strong and weak siRNA differ dramatically. Unlike therelatively strong values exhibited by siRNA in the >95% silencing group,siRNAs that exhibit poor silencing activity have weak AISP values (−7.6,−7.5, and −7.5 kcal/mol for positions 1, 2 and 3 respectively).

Overall the profiles of both strong and weak siRNAs form distinctsinusoidal shapes that are roughly 180° out-of-phase with each other.While these thermodynamic descriptions define the archetypal profile ofa strong siRNA, it will likely be the case that neither the ΔG valuesgiven for key positions in the profile or the absolute position of theprofile along the Y-axis (i.e. the ΔG-axis) are absolutes. Profiles thatare shifted upward or downward (i.e. having on an average, higher orlower values at every position) but retain the relative shape andposition of the profile along the X-axis can be foreseen as beingequally effective as the model profile described here. Moreover, it islikely that siRNA that have strong or even stronger gene-specificsilencing effects might have exaggerated ΔG values (either higher orlower) at key positions. Thus, for instance, it is possible that the5′-most position of the sense strand (position 19) could have ΔG valuesof 7.4 kcal/mol or lower and still be a strong siRNA if, for instance, aG-C→G-T/U mismatch were substituted at position 19 and altered duplexstability. Similarly, position 12 and position 7 could have values above8.3 kcal/mol and below 7.7 kcal/mole, respectively, without abating thesilencing effectiveness of the molecule. Thus, for instance, at position12, a stabilizing chemical modification (e.g. a chemical modification ofthe 2′ position of the sugar backbone) could be added that increases theaverage internal stability at that position. Similarly, at position 7,mismatches similar to those described previously could be introducedthat would lower the ΔG values at that position.

Lastly, it is important to note that while functional and non-functionalsiRNA were originally defined as those molecules having specificsilencing properties, both broader or more limiting parameters can beused to define these molecules. As used herein, unless otherwisespecified, “non-functional siRNA” are defined as those siRNA that induceless than 50% (<50%) target silencing, “semi-functional siRNA” induce50-79% target silencing, “functional siRNA” are molecules that induce80-95% gene silencing, and “highly-functional siRNA” are molecules thatinduce great than 95% gene silencing. These definitions are not intendedto be rigid and can vary depending upon the design and needs of theapplication. For instance, it is possible that a researcher attemptingto map a gene to a chromosome using a functional assay, may identify ansiRNA that reduces gene activity by only 30%. While this level of genesilencing may be “non-functional” for e.g. therapeutic needs, it issufficient for gene mapping purposes and is, under these uses andconditions, “functional.” For these reasons, functional siRNA can bedefined as those molecules having greater than 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% silencing capabilities at 100 nM transfectionconditions. Similarly, depending upon the needs of the study and/orapplication, non-functional and semi-functional siRNA can be defined ashaving different parameters. For instance, semi-functional siRNA can bedefined as being those molecules that induce 20%, 30%, 40%, 50%, 60%, or70% silencing at 100 nM transfection conditions. Similarly,non-functional siRNA can be defined as being those molecules thatsilence gene expression by less than 70%, 60%, 50%, 40%, 30%, or less.Nonetheless, unless otherwise stated, the descriptions stated in the“Definitions” section of this text should be applied.

Functional attributes can be assigned to each of the key positions inthe AISP of strong siRNA. The low 5′ (sense strand) AISP values ofstrong siRNAs may be necessary for determining which end of the moleculeenters the RISC complex. In contrast, the high and low AISP valuesobserved in the central regions of the molecule may be critical forsiRNA-target mRNA interactions and product release, respectively.

If the AISP values described above accurately define the thermodynamicparameters of strong siRNA, it would be expected that similar patternswould be observed in strong siRNA isolated from nature. Natural siRNAsexist in a harsh, RNase-rich environment and it can be hypothesized thatonly those siRNA that exhibit heightened affinity for RISC (i.e. siRNAthat exhibit an average internal stability profile similar to thoseobserved in strong siRNA) would survive in an intracellular environment.This hypothesis was tested using GFP-specific siRNA isolated from N.benthamiana. Llave et al. (2002) Endogenous and Silencing-AssociatedSmall RNAs in Plants, The Plant Cell 14, 1605-1619, introduced longdouble-stranded GFP-encoding RNA into plants and subsequentlyre-isolated GFP-specific siRNA from the tissues. The AISP of fifty-nineof these GFP-siRNA were determined, averaged, and subsequently plottedalongside the AISP profile obtained from the cyclophilinB/DBI/luciferase siRNA having >90% silencing properties (FIG. 6B).Comparison of the two groups show that profiles are nearly identical.This finding validates the information provided by the internalstability profiles and demonstrates that: (1) the profile identified byanalysis of the cyclophilin B/DBI/luciferase siRNAs are not genespecific; and (2) AISP values can be used to search for strong siRNAs ina variety of species.

Both chemical modifications and base-pair mismatches can be incorporatedinto siRNA to alter the duplex's AISP and functionality. For instance,introduction of mismatches at positions 1 or 2 of the sense stranddestabilized the 5′end of the sense strand and increases thefunctionality of the molecule (see Luc, FIG. 7). Similarly, addition of2′-O-methyl groups to positions 1 and 2 of the sense strand can alsoalter the AISP and (as a result) increase both the functionality of themolecule and eliminate off-target effects that results from sense strandhomology with the unrelated targets (FIGS. 8A, 8B).

Rationale for Criteria in a Biological Context

The fate of siRNA in the RNAi pathway may be described in 5 major steps:(1) duplex recognition and pre-RISC complex formation; (2) ATP-dependentduplex unwinding/strand selection and RISC activation; (3) mRNA targetidentification; (4) mRNA cleavage, and (5) product release (FIG. 1).Given the level of nucleic acid-protein interactions at each step, siRNAfunctionality is likely influenced by specific biophysical and molecularproperties that promote efficient interactions within the context of themulti-component complexes. Indeed, the systematic analysis of the siRNAtest set identified multiple factors that correlate well withfunctionality. When combined into a single algorithm, they proved to bevery effective in selecting active siRNAs.

The factors described here may also be predictive of key functionalassociations important for each step in RNAi. For example, the potentialformation of internal hairpin structures correlated negatively withsiRNA functionality. Complementary strands with stable internal repeatsare more likely to exist as stable hairpins thus decreasing theeffective concentration of the functional duplex form. This suggeststhat the duplex is the preferred conformation for initial pre-RISCassociation. Indeed, although single complementary strands can inducegene silencing, the effective concentration required is at least twoorders of magnitude higher than that of the duplex form.

siRNA-pre-RISC complex formation is followed by an ATP-dependent duplexunwinding step and “activation” of the RISC. The siRNA functionality wasshown to correlate with overall low internal stability of the duplex andlow internal stability of the 3′ sense end (or differential internalstability of the 3′ sense compare to the 5′ sense strand), which mayreflect strand selection and entry into the RISC. Overall duplexstability and low internal stability at the 3′ end of the sense strandwere also correlated with siRNA functionality. Interestingly, siRNAswith very high and very low overall stability profiles correlatestrongly with non-functional duplexes. One interpretation is that highinternal stability prevents efficient unwinding while very low stabilityreduces siRNA target affinity and subsequent mRNA cleavage by the RISC.

Several criteria describe base preferences at specific positions of thesense strand and are even more intriguing when considering theirpotential mechanistic roles in target recognition and mRNA cleavage.Base preferences for A at position 19 of the sense strand but not C, areparticularly interesting because they reflect the same base preferencesobserved for naturally occurring miRNA precursors. That is, among thereported miRNA precursor sequences 75% contain a U at position 1 whichcorresponds to an A in position 19 of the sense strand of siRNAs, whileG was under-represented in this same position for miRNA precursors.These observations support the hypothesis that both miRNA precursors andsiRNA duplexes are processed by very similar if not identical proteinmachinery. The functional interpretation of the predominance of a U/Abase pair is that it promotes flexibility at the 5′antisense ends ofboth siRNA duplexes and miRNA precursors and facilitates efficientunwinding and selective strand entrance into an activated RISC.

Among the criteria associated with base preferences that are likely toinfluence mRNA cleavage or possibly product release, the preference forU at position 10 of the sense strand exhibited the greatest impact,enhancing the probability of selecting an F80 sequence by 13.3%.Activated RISC preferentially cleaves target mRNA between nucleotides 10and 11 relative to the 5′ end of the complementary targeting strand.Therefore, it may be that U, the preferred base for mostendoribonucleases, at this position supports more efficient cleavage.Alternatively, a U/A by between the targeting siRNA strand and itscognate target mRNA may create an optimal conformation for theRISC-associated “slicing” activity.

Pooling

According to another embodiment, the present invention provides a poolof at least two siRNAs, preferably in the form of a kit or therapeuticreagent, wherein one strand of each of the siRNAs, the sense strandcomprises a sequence that is substantially similar to a sequence withina target mRNA. The opposite strand, the antisense strand, willpreferably comprise a sequence that is substantially complementary tothat of the target mRNA. More preferably, one strand of each siRNA willcomprise a sequence that is identical to a sequence that is contained inthe target mRNA. Most preferably, each siRNA will be 19 base pairs inlength, and one strand of each of the siRNAs will be 100% complementaryto a portion of the target mRNA.

By increasing the number of siRNAs directed to a particular target usinga pool or kit, one is able both to increase the likelihood that at leastone siRNA with satisfactory functionality will be included, as well asto benefit from additive or synergistic effects. Further, when two ormore siRNAs directed against a single gene do not have satisfactorylevels of functionality alone, if combined, they may satisfactorilypromote degradation of the target messenger RNA and successfully inhibittranslation. By including multiple siRNAs in the system, not only is theprobability of silencing increased, but the economics of operation arealso improved when compared to adding different siRNAs sequentially.This effect is contrary to the conventional wisdom that the concurrentuse of multiple siRNA will negatively impact gene silencing (e.g. Holen,T. et al. (2003) “Similar behavior of single strand and double strandsiRNAs suggests they act through a common RNAi pathway.” NAR 31:2401-21407).

In fact, when two siRNAs were pooled together, 54% of the pools of twosiRNAs induced more than 95% gene silencing. Thus, a 2.5-fold increasein the percentage of functionality was achieved by randomly combiningtwo siRNAs. Further, over 84% of pools containing two siRNAs inducedmore than 80% gene silencing.

More preferably, the kit is comprised of at least three siRNAs, whereinone strand of each siRNA comprises a sequence that is substantiallysimilar to a sequence of the target mRNA and the other strand comprisesa sequence that is substantially complementary to the region of thetarget mRNA. As with the kit that comprises at least two siRNAs, morepreferably one strand will comprise a sequence that is identical to asequence that is contained in the mRNA and another strand that is 100%complementary to a sequence that is contained in the mRNA. Duringexperiments, when three siRNAs were combined together, 60% of the poolsinduced more than 95% gene silencing and 92% of the pools induced morethan 80% gene silencing.

Further, even more preferably, the kit is comprised of at least foursiRNAs, wherein one strand of each siRNA comprises a sequence that issubstantially similar to a region of the sequence of the target mRNA,and the other strand comprises a sequence that is substantiallycomplementary to the region of the target mRNA. As with the kit or poolthat comprises at least two siRNAs, more preferably one strand of eachof the siRNA duplexes will comprise a sequence that is identical to asequence that is contained in the mRNA, and another strand that is 100%complementary to a sequence that is contained in the mRNA.

Additionally, kits and pools with at least five, at least six, and atleast seven siRNAs may also be useful with the present invention. Forexample, pools of five siRNA induced 95% gene silencing with 77%probability and 80% silencing with 98.8% probability. Thus, pooling ofsiRNAs together can result in the creation of a target-specificsilencing reagent with almost a 99% probability of being functional. Thefact that such high levels of success are achievable using such pools ofsiRNA, enables one to dispense with costly and time-consumingtarget-specific validation procedures.

For this embodiment, as well as the other aforementioned embodiments,each of the siRNAs within a pool will preferably comprise between 18 and30 base pairs, more preferably between 18 and 25 base pairs, and mostpreferably 19 base pairs. Within each siRNA, preferably at least 18contiguous bases of the antisense strand will be 100% complementary tothe target mRNA. More preferably, at least 19 contiguous bases of theantisense strand will be 100% complementary to the target mRNA.Additionally, there may be overhangs on either the sense strand or theantisense strand, and these overhangs may be at either the 5′ end or the3′ end of either of the strands, for example there may be one or moreoverhangs of 1-6 bases. When overhangs are present, they are notincluded in the calculation of the number of base pairs. The twonucleotide 3′ overhangs mimic natural siRNAs and are commonly used butare not essential. Preferably, the overhangs should consist of twonucleotides, most often dTdT or UU at the 3′ end of the sense andantisense strand that are not complementary to the target sequence. ThesiRNAs may be produced by any method that is now known or that comes tobe known for synthesizing double stranded RNA that one skilled in theart would appreciate would be useful in the present invention.Preferably, the siRNAs will be produced by Dharmacon's proprietary ACE®technology. However, other methods for synthesizing siRNAs are wellknown to persons skilled in the art and include, but are not limited to,any chemical synthesis of RNA oligonucleotides, ligation of shorteroligonucleotides, in vitro transcription of RNA oligonucleotides, theuse of vectors for expression within cells, recombinant Dicer productsand PCR products.

The siRNA duplexes within the aforementioned pools of siRNAs maycorrespond to overlapping sequences within a particular mRNA, ornon-overlapping sequences of the mRNA. However, preferably theycorrespond to non-overlapping sequences. Further, each siRNA may beselected randomly, or one or more of the siRNA may be selected accordingto the criteria discussed above for maximizing the effectiveness ofsiRNA.

Included in the definition of siRNAs are siRNAs that contain substitutedand/or labeled nucleotides that may, for example, be labeled byradioactivity, fluorescence or mass. The most common substitutions areat the 2′ position of the ribose sugar, where moieties such as H(hydrogen) F, NH₃, OCH₃ and other O-alkyl, alkenyl, alkynyl, andorthoesters, may be substituted, or in the phosphorous backbone, wheresulfur, amines or hydrocarbons may be substituted for the bridging ofnon-bridging atoms in the phosphodiester bond. Examples of modifiedsiRNAs are explained more fully in commonly assigned U.S. patentapplication Ser. No. 10/613,077, filed Jul. 1, 2003, which isincorporated by reference herein.

Additionally, as noted above, the cell type into which the siRNA isintroduced may affect the ability of the siRNA to enter the cell;however, it does not appear to affect the ability of the siRNA tofunction once it enters the cell. Methods for introducingdouble-stranded RNA into various cell types are well known to personsskilled in the art.

As persons skilled in the art are aware, in certain species, thepresence of proteins such as RdRP, the RNA-dependent RNA polymerase, maycatalytically enhance the activity of the siRNA. For example, RdRPpropagates the RNAi effect in C. elegans and other non-mammalianorganisms. In fact, in organisms that contain these proteins, the siRNAmay be inherited. Two other proteins that are well studied and known tobe a part of the machinery are members of the Argonaute family andDicer, as well as their homologues. There is also initial evidence thatthe RISC complex might be associated with the ribosome so the moreefficiently translated mRNAs will be more susceptible to silencing thanothers.

Another very important factor in the efficacy of siRNA is mRNAlocalization. In general, only cytoplasmic mRNAs are considered to beaccessible to RNAi to any appreciable degree. However, appropriatelydesigned siRNAs, for example, siRNAs modified with internucleotidelinkages, may be able to cause silencing by acting in the nucleus.Examples of these types of modifications are described in commonlyassigned U.S. patent application Ser. Nos. 10/431,027 and 10/613,077,each of which is incorporated by reference herein.

As described above, even when one selects at least two siRNAs at random,the effectiveness of the two may be greater than one would predict basedon the effectiveness of two individual siRNAs. This additive orsynergistic effect is particularly noticeable as one increases to atleast three siRNAs, and even more noticeable as one moves to at leastfour siRNAs. Surprisingly, the pooling of the non-functional andsemi-functional siRNAs, particularly more than five siRNAs, can lead toa silencing mixture that is as effective if not more effective than anyone particular functional siRNA.

Within the kit of the present invention, preferably each siRNA will bepresent in a concentration of between 0.001 and 200 μM, more preferablybetween 0.01 and 200 nM, and most preferably between 0.1 and 10 nM.

In addition to preferably comprising at least four or five siRNAs, thekit of the present invention will also preferably comprise a buffer tokeep the siRNA duplex stable. Persons skilled in the art are aware ofbuffers suitable for keeping siRNA stable. For example, the buffer maybe comprised of 100 mM KCl, 30 mM HEPES-pH 7.5, and 1 mM MgCl₂.Alternatively, kits might contain complementary strands that contain anyone of a number of chemical modifications (e.g. a 2′-O-ACE) that protectthe agents from degradation by nucleases. In this instance, the user may(or may not) remove the modifying protective group (e.g. deprotect)before annealing the two complementary strands together.

By way of example, the kit may be organized such that pools of siRNAduplexes are provided on an array or microarray of wells or drops for aparticular gene set or for unrelated genes. The array may, for example,be in 96 wells, 384 wells or 1284 wells arrayed in a plastic plate or ona glass slide using techniques now known or that come to be known topersons skilled in the art. Within an array, preferably there will becontrols such as functional anti-lamin A/C, cyclophilin and two siRNAduplexes that are not specific to the gene of interest.

In order to ensure stability of the siRNA pools prior to usage, they maybe retained in lyophilized form at minus twenty degrees (−20° C.) untilthey are ready for use. Prior to usage, they should be resuspended;however, even once resuspended, for example, in the aforementionedbuffer, they should be kept at minus twenty degrees, (−20° C.) untilused. The aforementioned buffer, prior to use, may be stored atapproximately 4° C. or room temperature. Effective temperatures at whichto conduct transfections are well known to persons skilled in the artand include for example, room temperature.

The kit may be applied either in vivo or in vitro. Preferably, the siRNAof the pools or kits is applied to a cell through transfection,employing standard transfection protocols. These methods are well knownto persons skilled in the art and include the use of lipid-basedcarriers, electroporation, cationic carriers, and microinjection.Further, one could apply the present invention by synthesizingequivalent DNA sequences (either as two separate, complementary strands,or as hairpin molecules) instead of siRNA sequences and introducing theminto cells through vectors. Once in the cells, the cloned DNA could betranscribed, thereby forcing the cells to generate the siRNA. Examplesof vectors suitable for use with the present application include but arenot limited to the standard transient expression vectors, adenoviruses,retroviruses, lentivirus-based vectors, as well as other traditionalexpression vectors. Any vector that has an adequate siRNA expression andprocession module may be used. Furthermore, certain chemicalmodifications to siRNAs, including but not limited to conjugations toother molecules, may be used to facilitate delivery. For certainapplications it may be preferable to deliver molecules withouttransfection by simply formulating in a physiological acceptablesolution.

This embodiment may be used in connection with any of the aforementionedembodiments. Accordingly, the sequences within any pool may be selectedby rational design.

Multigene Silencing

In addition to developing kits that contain multiple siRNA directedagainst a single gene, another embodiment includes the use of multiplesiRNA targeting multiple genes. Multiple genes may be targeted throughthe use of high- or hyper-functional siRNA. High- or hyper-functionalsiRNA that exhibit increased potency, require lower concentrations toinduce desired phenotypic (and thus therapeutic) effects. Thiscircumvents RISC saturation. It therefore reasons that if lowerconcentrations of a single siRNA are needed for knockout or knockdownexpression of one gene, then the remaining (uncomplexed) RISC will befree and available to interact with siRNA directed against two, three,four, or more, genes. Thus in this embodiment, the authors describe theuse of highly functional or hyper-functional siRNA to knock out threeseparate genes. More preferably, such reagents could be combined toknockout four distinct genes. Even more preferably, highly functional orhyperfunctional siRNA could be used to knock out five distinct genes.Most preferably, siRNA of this type could be used to knockout orknockdown the expression of six or more genes.

Hyperfunctional siRNA

The term hyperfunctional siRNA (hf-siRNA) describes a subset of thesiRNA population that induces RNAi in cells at low- or sub-nanomolarconcentrations for extended periods of time. These traits, heightenedpotency and extended longevity of the RNAi phenotype, are highlyattractive from a therapeutic standpoint. Agents having higher potencyrequire lesser amounts of the molecule to achieve the desiredphysiological response, thus reducing the probability of side effectsdue to “off-target” interference. In addition to the potentialtherapeutic benefits associated with hyperfunctional siRNA, hf-siRNA arealso desirable from an economic position. Hyperfunctional siRNA may costless on a per-treatment basis, thus reducing the overall expenditures toboth the manufacturer and the consumer.

Identification of hyperfunctional siRNA involves multiple steps that aredesigned to examine an individual siRNA agent's concentration- and/orlongevity-profiles. In one non-limiting example, a population of siRNAdirected against a single gene are first analyzed using the previouslydescribed algorithm (Formula VIII). Individual siRNA are then introducedinto a test cell line and assessed for the ability to degrade the targetmRNA. It is important to note that when performing this step it is notnecessary to test all of the siRNA. Instead, it is sufficient to testonly those siRNA having the highest SMARTSCORES™, or siRNA ranking (i.e.SMARTSCORE™>−10). Subsequently, the gene silencing data is plottedagainst the SMARTSCORES™, or siRNA rankings (see FIG. 9). siRNA that (1)induce a high degree of gene silencing (i.e. they induce greater than80% gene knockdown) and (2) have superior SMARTSCORES™, or siRNArankings (i.e. a SMARTSCORE™ of >−10, suggesting a desirable averageinternal stability profile) are selected for further investigationsdesigned to better understand the molecule's potency and longevity. Inone, non-limiting study dedicated to understanding a molecule's potency,an siRNA is introduced into one (or more) cell types in increasinglydiminishing concentrations (e.g. 3.0→0.3 nM). Subsequently, the level ofgene silencing induced by each concentration is examined and siRNA thatexhibit hyperfunctional potency (i.e. those that induce 80% silencing orgreater at e.g. picomolar concentrations) are identified. In a secondstudy, the longevity profiles of siRNA having high (>−10) SMARTSCORES™,or siRNA rankings, and greater than 80% silencing are examined. In onenon-limiting example of how this is achieved, siRNA are introduced intoa test cell line and the levels of RNAi are measured over an extendedperiod of time (e.g. 24-168 hrs). siRNAs that exhibit strong RNAinterference patterns (i.e. >80% interference) for periods of timegreater than, e.g., 120 hours are thus identified. Studies similar tothose described above can be performed on any and all of the >10⁶ siRNAincluded in this document to further define the most functional moleculefor any given gene. Molecules possessing one or both properties(extended longevity and heightened potency) are labeled “hyperfunctionalsiRNA,” and earmarked as candidates for future therapeutic studies.

While the example(s) given above describe one means by whichhyperfunctional siRNA can be isolated, neither the assays themselves northe selection parameters used are rigid and can vary with each family ofsiRNA. Families of siRNA include siRNAs directed against a single gene,or directed against a related family of genes.

The highest quality siRNA achievable for any given gene may varyconsiderably. Thus, for example, in the case of one gene (gene X),rigorous studies such as those described above may enable theidentification of an siRNA that, at picomolar concentrations, induces99⁺% silencing for a period of 10 days. Yet identical studies of asecond gene (gene Y) may yield an siRNA that at high nanomolarconcentrations (e.g. 100 nM) induces only 75% silencing for a period of2 days. Both molecules represent the very optimum siRNA for theirrespective gene targets and therefore are designated “hyperfunctional.”Yet due to a variety of factors including but not limited to targetconcentration, siRNA stability, cell type, off-target interference, andothers, equivalent levels of potency and longevity are not achievable.Thus, for these reasons, the parameters described in the beforementioned assays, can vary. While the initial screen selected siRNA thathad SMARTSCORES™, or siRNA rankings, above −10 and a gene silencingcapability of greater than 80%, selections that have stronger (orweaker) parameters can be implemented. Similarly, in the subsequentstudies designed to identify molecules with high potency and longevity,the desired cutoff criteria (i.e. the lowest concentration that inducesa desirable level of interference, or the longest period of time thatinterference can be observed) can vary. The experimentation subsequentto application of the rational criteria of this application issignificantly reduced where one is trying to obtain a suitablehyperfunctional siRNA for, for example, therapeutic use. When, forexample, the additional experimentation of the type described herein isapplied by one skilled in the art with this disclosure in hand, ahyperfunctional siRNA is readily identified.

The siRNA may be introduced into a cell by any method that is now knownor that comes to be known and that from reading this disclosure, personsskilled in the art would determine would be useful in connection withthe present invention in enabling siRNA to cross the cellular membrane.These methods include, but are not limited to, any manner oftransfection, such as for example transfection employing DEAE-Dextran,calcium phosphate, cationic lipids/liposomes, micelles, manipulation ofpressure, microinjection, electroporation, immunoporation, use ofvectors such as viruses, plasmids, cosmids, bacteriophages, cellfusions, and coupling of the polynucleotides to specific conjugates orligands such as antibodies, antigens, or receptors, passiveintroduction, adding moieties to the siRNA that facilitate its uptake,and the like.

Having described the invention with a degree of particularity, exampleswill now be provided. These examples are not intended to and should notbe construed to limit the scope of the claims in any way.

Examples General Techniques and Nomenclatures

siRNA nomenclature. All siRNA duplexes are referred to by sense strand.The first nucleotide of the 5′-end of the sense strand is position 1,which corresponds to position 19 of the antisense strand for a 19-mer.In most cases, to compare results from different experiments, silencingwas determined by measuring specific transcript mRNA levels or enzymaticactivity associated with specific transcript levels, 24 hourspost-transfection, with siRNA concentrations held constant at 100 nM.For all experiments, unelss otherwise specified transfection efficiencywas ensured to be over 95%, and no detectable cellular toxicity wasobserved. The following system of nomenclature was used to compare andreport siRNA-silencing functionality: “F” followed by the degree ofminimal knockdown. For example, F50 signifies at least 50% knockdown,F80 means at least 80%, and so forth. For this study, all sub-F50 siRNAswere considered non-functional.

Cell culture and transfection. 96-well plates are coated with 50 μl of50 mg/ml poly-L-lysine (Sigma) for 1 hr, and then washed 3× withdistilled water before being dried for 20 min. HEK293 cells orHEK293Lucs or any other cell type of interest are released from theirsolid support by trypsinization, diluted to 3.5×10⁵ cells/ml, followedby the addition of 100 μL of cells/well. Plates are then incubatedovernight at 37° C., 5% CO₂. Transfection procedures can vary widelydepending on the cell type and transfection reagents. In onenon-limiting example, a transfection mixture consisting of 2 mL Opti-MEMI (Gibco-BRL), 80 μl Lipofectamine 2000 (Invitrogen), 15 μL SUPERNasinat 20 U/μl (Ambion), and 1.5 μl of reporter gene plasmid at 1 μg/μl isprepared in 5-ml polystyrene round bottom tubes. 100 μl of transfectionreagent is then combined with 100 μl of siRNAs in polystyrene deep-welltiter plates (Beckman) and incubated for 20 to 30 min at room temp. 550μl of Opti-MEM is then added to each well to bring the final siRNAconcentration to 100 nM. Plates are then sealed with parafilm and mixed.Media is removed from HEK293 cells and replaced with 95 μl oftransfection mixture. Cells are incubated overnight at 37° C., 5% CO₂.

Quantification of gene knockdown. A variety of quantification procedurescan be used to measure the level of silencing induced by siRNA or siRNApools. In one non-limiting example: to measure mRNA levels 24 hrspost-transfection, QuantiGene branched-DNA (bDNA) kits (Bayer) (Wang, etal, Regulation of insulin preRNA splicing by glucose. Proc Natl Acad Sci1997, 94:4360.) are used according to manufacturer instructions. Tomeasure luciferase activity, media is removed from HEK293 cells 24 hrspost-transfection, and 50 μl of Steady-GLO reagent (Promega) is added.After 5 min, plates are analyzed on a plate reader.

Example I Sequences Used to Develop the Algorithm

Anti-Firefly and anti-Cyclophilin siRNAs panels (FIGS. 5A, B) sortedaccording to using Formula VIII predicted values. All siRNAs scoringmore than 0 (formula VIII) and more then 20 (formula IX) are fullyfunctional. All ninety sequences for each gene (and DBI) appear below inTable IV.

TABLE IV Cyclo 1 SEQ ID NO 032 GUUCCAAAAACAGUGGAUA Cyclo 2 SEQ ID NO 033UCCAAAAACAGUGGAUAAU Cyclo 3 SEQ ID NO 034 CAAAAACAGUGGAUAAUUU Cyclo 4SEQ ID NO 035 AAAACAGUGGAUAAUUUUG Cyclo 5 SEQ ID NO 036AACAGUGGAUAAUUUUGUG Cyclo 6 SEQ ID NO 037 CAGUGGAUAAUUUUGUGGC Cyclo 7SEQ ID NO 038 GUGGAUAAUUUUGUGGCCU Cyclo 8 SEQ ID NO 039GGAUAAUUUUGUGGCCUUA Cyclo 9 SEQ ID NO 040 AUAAUUUUGUGGCCUUAGC Cyclo 10SEQ ID NO 041 AAUUUUGUGGCCUUAGCUA Cyclo 11 SEQ ID NO 042UUUUGUGGCCUUAGCUACA Cyclo 12 SEQ ID NO 043 UUGUGGCCUUAGCUACAGG Cyclo 13SEQ ID NO 044 GUGGCCUUAGCUACAGGAG Cyclo 14 SEQ ID NO 045GGCCUUAGCUACAGGAGAG Cyclo 15 SEQ ID NO 046 CCUUAGCUACAGGAGAGAA Cyclo 16SEQ ID NO 047 UUAGCUACAGGAGAGAAAG Cyclo 17 SEQ ID NO 048AGCUACAGGAGAGAAAGGA Cyclo 18 SEQ ID NO 049 CUACAGGAGAGAAAGGAUU Cyclo 19SEQ ID NO 050 ACAGGAGAGAAAGGAUUUG Cyclo 20 SEQ ID NO 051AGGAGAGAAAGGAUUUGGC Cyclo 21 SEQ ID NO 052 GAGAGAAAGGAUUUGGCUA Cyclo 22SEQ ID NO 053 GAGAAAGGAUUUGGCUACA Cyclo 23 SEQ ID NO 054GAAAGGAUUUGGCUACAAA Cyclo 24 SEQ ID NO 055 AAGGAUUUGGCUACAAAAA Cyclo 25SEQ ID NO 056 GGAUUUGGCUACAAAAACA Cyclo 26 SEQ ID NO 057AUUUGGCUACAAAAACAGC Cyclo 27 SEQ ID NO 058 UUGGCUACAAAAACAGCAA Cyclo 28SEQ ID NO 059 GGCUACAAAAACAGCAAAU Cyclo 29 SEQ ID NO 060CUACAAAAACAGCAAAUUC Cyclo 30 SEQ ID NO 061 ACAAAAACAGCAAAUUCCA Cyclo 31SEQ ID NO 062 AAAAACAGCAAAUUCCAUC Cyclo 32 SEQ ID NO 063AAACAGCAAAUUCCAUCGU Cyclo 33 SEQ ID NO 064 ACAGCAAAUUCCAUCGUGU Cyclo 34SEQ ID NO 065 AGCAAAUUCCAUCGUGUAA Cyclo 35 SEQ ID NO 066CAAAUUCCAUCGUGUAAUC Cyclo 36 SEQ ID NO 067 AAUUCCAUCGUGUAAUCAA Cyclo 37SEQ ID NO 068 UUCCAUCGUGUAAUCAAGG Cyclo 38 SEQ ID NO 069CCAUCGUGUAAUCAAGGAC Cyclo 39 SEQ ID NO 070 AUCGUGUAAUCAAGGACUU Cyclo 40SEQ ID NO 071 CGUGUAAUCAAGGACUUCA Cyclo 41 SEQ ID NO 072UGUAAUCAAGGACUUCAUG Cyclo 42 SEQ ID NO 073 UAAUCAAGGACUUCAUGAU Cyclo 43SEQ ID NO 074 AUCAAGGACUUCAUGAUCC Cyclo 44 SEQ ID NO 075CAAGGACUUCAUGAUCCAG Cyclo 45 SEQ ID NO 076 AGGACUUCAUGAUCCAGGG Cyclo 46SEQ ID NO 077 GACUUCAUGAUCCAGGGCG Cyclo 47 SEQ ID NO 078CUUCAUGAUCCAGGGCGGA Cyclo 48 SEQ ID NO 079 UCAUGAUCCAGGGCGGAGA Cyclo 49SEQ ID NO 080 AUGAUCCAGGGCGGAGACU Cyclo 50 SEQ ID NO 081GAUCCAGGGCGGAGACUUC Cyclo 51 SEQ ID NO 082 UCCAGGGCGGAGACUUCAC Cyclo 52SEQ ID NO 083 CAGGGCGGAGACUUCACCA Cyclo 53 SEQ ID NO 084GGGCGGAGACUUCACCAGG Cyclo 54 SEQ ID NO 085 GCGGAGACUUCACCAGGGG Cyclo 55SEQ ID NO 086 GGAGACUUCACCAGGGGAG Cyclo 56 SEQ ID NO 087AGACUUCACCAGGGGAGAU Cyclo 57 SEQ ID NO 088 ACUUCACCAGGGGAGAUGG Cyclo 58SEQ ID NO 089 UUCACCAGGGGAGAUGGCA Cyclo 59 SEQ ID NO 090CACCAGGGGAGAUGGCACA Cyclo 60 SEQ ID NO 091 CCAGGGGAGAUGGCACAGG Cyclo 61SEQ ID NO 092 AGGGGAGAUGGCACAGGAG Cyclo 62 SEQ ID NO 093GGGAGAUGGCACAGGAGGA Cyclo 63 SEQ ID NO 094 GAGAUGGCACAGGAGGAAA Cyclo 64SEQ ID NO 095 GAUGGCACAGGAGGAAAGA Cyclo 65 SEQ ID NO 096UGGCACAGGAGGAAAGAGC Cyclo 66 SEQ ID NO 097 GCACAGGAGGAAAGAGCAU Cyclo 67SEQ ID NO 098 ACAGGAGGAAAGAGCAUCU Cyclo 68 SEQ ID NO 099AGGAGGAAAGAGCAUCUAC Cyclo 69 SEQ ID NO 100 GAGGAAAGAGCAUCUACGG Cyclo 70SEQ ID NO 101 GGAAAGAGCAUCUACGGUG Cyclo 71 SEQ ID NO 102AAAGAGCAUCUACGGUGAG Cyclo 72 SEQ ID NO 103 AGAGCAUCUACGGUGAGCG Cyclo 73SEQ ID NO 104 AGCAUCUACGGUGAGCGCU Cyclo 74 SEQ ID NO 105CAUCUACGGUGAGCGCUUC Cyclo 75 SEQ ID NO 106 UCUACGGUGAGCGCUUCCC Cyclo 76SEQ ID NO 107 UACGGUGAGCGCUUCCCCG Cyclo 77 SEQ ID NO 108CGGUGAGCGCUUCCCCGAU Cyclo 78 SEQ ID NO 109 GUGAGCGCUUCCCCGAUGA Cyclo 79SEQ ID NO 110 GAGCGCUUCCCCGAUGAGA Cyclo 80 SEQ ID NO 111GCGCUUCCCCGAUGAGAAC Cyclo 81 SEQ ID NO 112 GCUUCCCCGAUGAGAACUU Cyclo 82SEQ ID NO 113 UUCCCCGAUGAGAACUUCA Cyclo 83 SEQ ID NO 114CCCCGAUGAGAACUUCAAA Cyclo 84 SEQ ID NO 115 CCGAUGAGAACUUCAAACU Cyclo 85SEQ ID NO 116 GAUGAGAACUUCAAACUGA Cyclo 86 SEQ ID NO 117UGAGAACUUCAAACUGAAG Cyclo 87 SEQ ID NO 118 AGAACUUCAAACUGAAGCA Cyclo 88SEQ ID NO 119 AACUUCAAACUGAAGCACU Cyclo 89 SEQ ID NO 120CUUCAAACUGAAGCACUAC Cyclo 90 SEQ ID NO 121 UCAAACUGAAGCACUACGG DB 1SEQ ID NO 122 ACGGGCAAGGCCAAGUGGG DB 2 SEQ ID NO 123 CGGGCAAGGCCAAGUGGGADB 3 SEQ ID NO 124 GGGCAAGGCCAAGUGGGAU DB 4 SEQ ID NO 125GGCAAGGCCAAGUGGGAUG DB 5 SEQ ID NO 126 GCAAGGCCAAGUGGGAUGC DB 6SEQ ID NO 127 CAAGGCCAAGUGGGAUGCC DB 7 SEQ ID NO 128 AAGGCCAAGUGGGAUGCCUDB 8 SEQ ID NO 129 AGGCCAAGUGGGAUGCCUG DB 9 SEQ ID NO 130GGCCAAGUGGGAUGCCUGG DB 10 SEQ ID NO 131 GCCAAGUGGGAUGCCUGGA DB 11SEQ ID NO 132 CCAAGUGGGAUGCCUGGAA DB 12 SEQ ID NO 133CAAGUGGGAUGCCUGGAAU DB 13 SEQ ID NO 134 AAGUGGGAUGCCUGGAAUG DB 14SEQ ID NO 135 AGUGGGAUGCCUGGAAUGA DB 15 SEQ ID NO 136GUGGGAUGCCUGGAAUGAG DB 16 SEQ ID NO 137 UGGGAUGCCUGGAAUGAGC DB 17SEQ ID NO 138 GGGAUGCCUGGAAUGAGCU DB 18 SEQ ID NO 139GGAUGCCUGGAAUGAGCUG DB 19 SEQ ID NO 140 GAUGCCUGGAAUGAGCUGA DB 20SEQ ID NO 141 AUGCCUGGAAUGAGCUGAA DB 21 SEQ ID NO 142UGCCUGGAAUGAGCUGAAA DB 22 SEQ ID NO 143 GCCUGGAAUGAGCUGAAAG DB 23SEQ ID NO 144 CCUGGAAUGAGCUGAAAGG DB 24 SEQ ID NO 145CUGGAAUGAGCUGAAAGGG DB 25 SEQ ID NO 146 UGGAAUGAGCUGAAAGGGA DB 26SEQ ID NO 147 GGAAUGAGCUGAAAGGGAC DB 27 SEQ ID NO 148GAAUGAGCUGAAAGGGACU DB 28 SEQ ID NO 149 AAUGAGCUGAAAGGGACUU DB 29SEQ ID NO 150 AUGAGCUGAAAGGGACUUC DB 30 SEQ ID NO 151UGAGCUGAAAGGGACUUCC DB 31 SEQ ID NO 152 GAGCUGAAAGGGACUUCCA DB 32SEQ ID NO 153 AGCUGAAAGGGACUUCCAA DB 33 SEQ ID NO 154GCUGAAAGGGACUUCCAAG DB 34 SEQ ID NO 155 CUGAAAGGGACUUCCAAGG DB 35SEQ ID NO 156 UGAAAGGGACUUCCAAGGA DB 36 SEQ ID NO 157GAAAGGGACUUCCAAGGAA DB 37 SEQ ID NO 158 AAAGGGACUUCCAAGGAAG DB 38SEQ ID NO 159 AAGGGACUUCCAAGGAAGA DB 39 SEQ ID NO 160AGGGACUUCCAAGGAAGAU DB 40 SEQ ID NO 161 GGGACUUCCAAGGAAGAUG DB 41SEQ ID NO 162 GGACUUCCAAGGAAGAUGC DB 42 SEQ ID NO 163GACUUCCAAGGAAGAUGCC DB 43 SEQ ID NO 164 ACUUCCAAGGAAGAUGCCA DB 44SEQ ID NO 165 CUUCCAAGGAAGAUGCCAU DB 45 SEQ ID NO 166UUCCAAGGAAGAUGCCAUG DB 46 SEQ ID NO 167 UCCAAGGAAGAUGCCAUGA DB 47SEQ ID NO 168 CCAAGGAAGAUGCCAUGAA DB 48 SEQ ID NO 169CAAGGAAGAUGCCAUGAAA DB 49 SEQ ID NO 170 AAGGAAGAUGCCAUGAAAG DB 50SEQ ID NO 171 AGGAAGAUGCCAUGAAAGC DB 51 SEQ ID NO 172GGAAGAUGCCAUGAAAGCU DB 52 SEQ ID NO 173 GAAGAUGCCAUGAAAGCUU DB 53SEQ ID NO 174 AAGAUGCCAUGAAAGCUUA DB 54 SEQ ID NO 175AGAUGCCAUGAAAGCUUAC DB 55 SEQ ID NO 176 GAUGCCAUGAAAGCUUACA DB 56SEQ ID NO 177 AUGCCAUGAAAGCUUACAU DB 57 SEQ ID NO 178UGCCAUGAAAGCUUACAUC DB 58 SEQ ID NO 179 GCCAUGAAAGCUUACAUCA DB 59SEQ ID NO 180 CCAUGAAAGCUUACAUCAA DB 60 SEQ ID NO 181CAUGAAAGCUUACAUCAAC DB 61 SEQ ID NO 182 AUGAAAGCUUACAUCAACA DB 62SEQ ID NO 183 UGAAAGCUUACAUCAACAA DB 63 SEQ ID NO 184GAAAGCUUACAUCAACAAA DB 64 SEQ ID NO 185 AAAGCUUACAUCAACAAAG DB 65SEQ ID NO 186 AAGCUUACAUCAACAAAGU DB 66 SEQ ID NO 187AGCUUACAUCAACAAAGUA DB 67 SEQ ID NO 188 GCUUACAUCAACAAAGUAG DB 68SEQ ID NO 189 CUUACAUCAACAAAGUAGA DB 69 SEQ ID NO 190UUACAUCAACAAAGUAGAA DB 70 SEQ ID NO 191 UACAUCAACAAAGUAGAAG DB 71SEQ ID NO 192 ACAUCAACAAAGUAGAAGA DB 72 SEQ ID NO 193CAUCAACAAAGUAGAAGAG DB 73 SEQ ID NO 194 AUCAACAAAGUAGAAGAGC DB 74SEQ ID NO 195 UCAACAAAGUAGAAGAGCU DB 75 SEQ ID NO 196CAACAAAGUAGAAGAGCUA DB 76 SEQ ID NO 197 AACAAAGUAGAAGAGCUAA DB 77SEQ ID NO 198 ACAAAGUAGAAGAGCUAAA DB 78 SEQ ID NO 199CAAAGUAGAAGAGCUAAAG DB 79 SEQ ID NO 200 AAAGUAGAAGAGCUAAAGA DB 80SEQ ID NO 201 AAGUAGAAGAGCUAAAGAA DB 81 SEQ ID NO 202AGUAGAAGAGCUAAAGAAA DB 82 SEQ ID NO 203 GUAGAAGAGCUAAAGAAAA DB 83SEQ ID NO 204 UAGAAGAGCUAAAGAAAAA DB 84 SEQ ID NO 205AGAAGAGCUAAAGAAAAAA DB 85 SEQ ID NO 206 GAAGAGCUAAAGAAAAAAU DB 86SEQ ID NO 207 AAGAGCUAAAGAAAAAAUA DB 87 SEQ ID NO 208AGAGCUAAAGAAAAAAUAC DB 88 SEQ ID NO 209 GAGCUAAAGAAAAAAUACG DB 89SEQ ID NO 210 AGCUAAAGAAAAAAUACGG DB 90 SEQ ID NO 211GCUAAAGAAAAAAUACGGG Luc 1 SEQ ID NO 212 AUCCUCAUAAAGGCCAAGA Luc 2SEQ ID NO 213 AGAUCCUCAUAAAGGCCAA Luc 3 SEQ ID NO 214AGAGAUCCUCAUAAAGGCC Luc 4 SEQ ID NO 215 AGAGAGAUCCUCAUAAAGG Luc 5SEQ ID NO 216 UCAGAGAGAUCCUCAUAAA Luc 6 SEQ ID NO 217AAUCAGAGAGAUCCUCAUA Luc 7 SEQ ID NO 218 AAAAUCAGAGAGAUCCUCA Luc 8SEQ ID NO 219 GAAAAAUCAGAGAGAUCCU Luc 9 SEQ ID NO 220AAGAAAAAUCAGAGAGAUC Luc 10 SEQ ID NO 221 GCAAGAAAAAUCAGAGAGA Luc 11SEQ ID NO 222 ACGCAAGAAAAAUCAGAGA Luc 12 SEQ ID NO 223CGACGCAAGAAAAAUCAGA Luc 13 SEQ ID NO 224 CUCGACGCAAGAAAAAUCA Luc 14SEQ ID NO 225 AACUCGACGCAAGAAAAAU Luc 15 SEQ ID NO 226AAAACUCGACGCAAGAAAA Luc 16 SEQ ID NO 227 GGAAAACUCGACGCAAGAA Luc 17SEQ ID NO 228 CCGGAAAACUCGACGCAAG Luc 18 SEQ ID NO 229UACCGGAAAACUCGACGCA Luc 19 SEQ ID NO 230 CUUACCGGAAAACUCGACG Luc 20SEQ ID NO 231 GUCUUACCGGAAAACUCGA Luc 21 SEQ ID NO 232AGGUCUUACCGGAAAACUC Luc 22 SEQ ID NO 233 AAAGGUCUUACCGGAAAAC Luc 23SEQ ID NO 234 CGAAAGGUCUUACCGGAAA Luc 24 SEQ ID NO 235ACCGAAAGGUCUUACCGGA Luc 25 SEQ ID NO 236 GUACCGAAAGGUCUUACCG Luc 26SEQ ID NO 237 AAGUACCGAAAGGUCUUAC Luc 27 SEQ ID NO 238CGAAGUACCGAAAGGUCUU Luc 28 SEQ ID NO 239 GACGAAGUACCGAAAGGUC Luc 29SEQ ID NO 240 UGGACGAAGUACCGAAAGG Luc 30 SEQ ID NO 241UGUGGACGAAGUACCGAAA Luc 31 SEQ ID NO 242 UUUGUGGACGAAGUACCGA Luc 32SEQ ID NO 243 UGUUUGUGGACGAAGUACC Luc 33 SEQ ID NO 244UGUGUUUGUGGACGAAGUA Luc 34 SEQ ID NO 245 GUUGUGUUUGUGGACGAAG Luc 35SEQ ID NO 246 GAGUUGUGUUUGUGGACGA Luc 36 SEQ ID NO 247AGGAGUUGUGUUUGUGGAC Luc 37 SEQ ID NO 248 GGAGGAGUUGUGUUUGUGG Luc 38SEQ ID NO 249 GCGGAGGAGUUGUGUUUGU Luc 39 SEQ ID NO 250GCGCGGAGGAGUUGUGUUU Luc 40 SEQ ID NO 251 UUGCGCGGAGGAGUUGUGU Luc 41SEQ ID NO 252 AGUUGCGCGGAGGAGUUGU Luc 42 SEQ ID NO 253AAAGUUGCGCGGAGGAGUU Luc 43 SEQ ID NO 254 AAAAAGUUGCGCGGAGGAG Luc 44SEQ ID NO 255 CGAAAAAGUUGCGCGGAGG Luc 45 SEQ ID NO 256CGCGAAAAAGUUGCGCGGA Luc 46 SEQ ID NO 257 ACCGCGAAAAAGUUGCGCG Luc 47SEQ ID NO 258 CAACCGCGAAAAAGUUGCG Luc 48 SEQ ID NO 259AACAACCGCGAAAAAGUUG Luc 49 SEQ ID NO 260 GUAACAACCGCGAAAAAGU Luc 50SEQ ID NO 261 AAGUAACAACCGCGAAAAA Luc 51 SEQ ID NO 262UCAAGUAACAACCGCGAAA Luc 52 SEQ ID NO 263 AGUCAAGUAACAACCGCGA Luc 53SEQ ID NO 264 CCAGUCAAGUAACAACCGC Luc 54 SEQ ID NO 265CGCCAGUCAAGUAACAACC Luc 55 SEQ ID NO 266 GUCGCCAGUCAAGUAACAA Luc 56SEQ ID NO 267 ACGUCGCCAGUCAAGUAAC Luc 57 SEQ ID NO 268UUACGUCGCCAGUCAAGUA Luc 58 SEQ ID NO 269 GAUUACGUCGCCAGUCAAG Luc 59SEQ ID NO 270 UGGAUUACGUCGCCAGUCA Luc 60 SEQ ID NO 271CGUGGAUUACGUCGCCAGU Luc 61 SEQ ID NO 272 AUCGUGGAUUACGUCGCCA Luc 62SEQ ID NO 273 AGAUCGUGGAUUACGUCGC Luc 63 SEQ ID NO 274AGAGAUCGUGGAUUACGUC Luc 64 SEQ ID NO 275 AAAGAGAUCGUGGAUUACG Luc 65SEQ ID NO 276 AAAAAGAGAUCGUGGAUUA Luc 66 SEQ ID NO 277GGAAAAAGAGAUCGUGGAU Luc 67 SEQ ID NO 278 ACGGAAAAAGAGAUCGUGG Luc 68SEQ ID NO 279 UGACGGAAAAAGAGAUCGU Luc 69 SEQ ID NO 280GAUGACGGAAAAAGAGAUC Luc 70 SEQ ID NO 281 ACGAUGACGGAAAAAGAGA Luc 71SEQ ID NO 282 AGACGAUGACGGAAAAAGA Luc 72 SEQ ID NO 283AAAGACGAUGACGGAAAAA Luc 73 SEQ ID NO 284 GGAAAGACGAUGACGGAAA Luc 74SEQ ID NO 285 ACGGAAAGACGAUGACGGA Luc 75 SEQ ID NO 286GCACGGAAAGACGAUGACG Luc 76 SEQ ID NO 287 GAGCACGGAAAGACGAUGA Luc 77SEQ ID NO 288 UGGAGCACGGAAAGACGAU Luc 78 SEQ ID NO 289UUUGGAGCACGGAAAGACG Luc 79 SEQ ID NO 290 GUUUUGGAGCACGGAAAGA Luc 80SEQ ID NO 291 UUGUUUUGGAGCACGGAAA Luc 81 SEQ ID NO 292UGUUGUUUUGGAGCACGGA Luc 82 SEQ ID NO 293 GUUGUUGUUUUGGAGCACG Luc 83SEQ ID NO 294 CCGUUGUUGUUUUGGAGCA Luc 84 SEQ ID NO 295CGCCGUUGUUGUUUUGGAG Luc 85 SEQ ID NO 296 GCCGCCGUUGUUGUUUUGG Luc 86SEQ ID NO 297 CCGCCGCCGUUGUUGUUUU Luc 87 SEQ ID NO 298UCCCGCCGCCGUUGUUGUU Luc 88 SEQ ID NO 299 CUUCCCGCCGCCGUUGUUG Luc 89SEQ ID NO 300 AACUUCCCGCCGCCGUUGU Luc 90 SEQ ID NO 301UGAACUUCCCGCCGCCGUU

Example II Validation of the Algorithm Using DBI, Luciferase, PLK, EGFR,and SEAP

The algorithm (Formula VIII) identified siRNAs for five genes, humanDBI, firefly luciferase (fLuc), renilla luciferase (rLuc), human PLK,and human secreted alkaline phosphatase (SEAP). Four individual siRNAswere selected on the basis of their SMARTSCORES™, or siRNA rankings,derived by analysis of their sequence using Formula VIII (all of thesiRNAs would be selected with Formula IX as well) and analyzed for theirability to silence their targets' expression. In addition to thescoring, a BLAST search was conducted for each siRNA. To minimize thepotential for off-target silencing effects, only those target sequenceswith more than three mismatches against unrelated sequences wereselected. Semizarov, et al (2003) Specificity of short interfering RNAdetermined through gene expression signatures. Proc. Natl. Acad. Sci.USA 100:6347. These duplexes were analyzed individually and in pools of4 and compared with several siRNAs that were randomly selected. Thefunctionality was measured a percentage of targeted gene knockdown ascompared to controls. All siRNAs were transfected as described by themethods above at 100 nM concentration into HEK293 using Lipofectamine2000. The level of the targeted gene expression was evaluated by B-DNAas described above and normalized to the non-specific control. FIG. 10shows that the siRNAs selected by the algorithm disclosed herein weresignificantly more potent than randomly selected siRNAs. The algorithmincreased the chances of identifying an F50 siRNA from 48% to 91%, andan F80 siRNA from 13% to 57%. In addition, pools of SMART siRNA silencethe selected target better than randomly selected pools (see FIG. 10F).

Example III Validation of the Algorithm Using Genes Involved inClathrin-Dependent Endocytosis

Components of clathrin-mediated endocytosis pathway are key tomodulating intracellular signaling and play important roles in disease.Chromosomal rearrangements that result in fusion transcripts between theMixed-Lineage Leukemia gene (MLL) and CALM (Clathrin assembly lymphoidmyeloid leukemia gene) are believed to play a role in leukemogenesis.Similarly, disruptions in Rab7 and Rab9, as well as HIP1(Huntingtin-interacting protein), genes that are believed to be involvedin endocytosis, are potentially responsible for ailments resulting inlipid storage, and neuronal diseases, respectively. For these reasons,siRNA directed against clathrin and other genes involved in theclathrin-mediated endocytotic pathway are potentially important researchand therapeutic tools.

siRNAs directed against genes involved in the clathrin-mediatedendocytosis pathways were selected using Formula VIII. The targetedgenes were clathrin heavy chain (CHC, accession #NM_(—)004859), clathrinlight chain A (CLCa, NM_(—)001833), clathrin light chain B (CLCb,NM_(—)001834), CALM (U45976), β2 subunit of AP-2 (β2, NM_(—)001282),Eps15 (NM_(—)001981), Eps15R (NM_(—)021235), dynamin II (DYNII,NM_(—)004945), Rab5a (BC001267), Rab5b (NM_(—)002868), Rab5c (AF141304),and EEA.1 (XM_(—)018197).

For each gene, four siRNAs duplexes with the highest scores wereselected and a BLAST search was conducted for each of them using theHuman EST database. In order to minimize the potential for off-targetsilencing effects, only those sequences with more than three mismatchesagainst un-related sequences were used. All duplexes were synthesized atDharmacon, Inc. as 21-mers with 3′-UU overhangs using a modified methodof 2′-ACE chemistry Scaringe (2000) Advanced 5′-silyl-2′-orthoesterapproach to RNA oligonucleotide synthesis, Methods Enzymol 317:3 and theantisense strand was chemically phosphorylated to insure maximizedactivity.

HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum, antibiotics and glutamine. siRNAduplexes were resuspended in 1× siRNA Universal buffer (Dharmacon, Inc.)to 20 μM prior to transfection. HeLa cells in 12-well plates weretransfected twice with 4 μl of 20 μM siRNA duplex in 3 μl Lipofectamine2000 reagent (Invitrogen, Carlsbad, Calif., USA) at 24-hour intervals.For the transfections in which 2 or 3 siRNA duplexes were included, theamount of each duplex was decreased, so that the total amount was thesame as in transfections with single siRNAs. Cells were plated intonormal culture medium 12 hours prior to experiments, and protein levelswere measured 2 or 4 days after the first transfection.

Equal amounts of lysates were resolved by electrophoresis, blotted, andstained with the antibody specific to targeted protein, as well asantibodies specific to unrelated proteins, PP1 phosphatase and Tsg101(not shown). The cells were lysed in Triton X-100/glycerolsolubilization buffer as described previously. Tebar, Bohlander, &Sorkin (1999) Clathrin Assembly Lymphoid Myeloid Leukemia (CALM)Protein: Localization in Endocytic-coated Pits, Interactions withClathrin, and the Impact of Overexpression on Clathrin-mediated Traffic,Mol. Biol. Cell 10:2687. Cell lysates were electrophoresed, transferredto nitrocellulose membranes, and Western blotting was performed withseveral antibodies followed by detection using enhancedchemiluminescence system (Pierce, Inc). Several x-ray films wereanalyzed to determine the linear range of the chemiluminescence signals,and the quantifications were performed using densitometry andAlphalmager v5.5 software (Alpha Innotech Corporation). In experimentswith Eps15R-targeted siRNAs, cell lysates were subjected toimmunoprecipitation with Ab860, and Eps15R was detected inimmunoprecipitates by Western blotting as described above.

The antibodies to assess the levels of each protein by Western blot wereobtained from the following sources: monoclonal antibody to clathrinheavy chain (TD.1) was obtained from American Type Culture Collection(Rockville, Md., USA); polyclonal antibody to dynamin II was obtainedfrom Affinity Bioreagents, Inc. (Golden, Colo., USA); monoclonalantibodies to EEA.1 and Rab5a were purchased from BD TransductionLaboratories (Los Angeles, Calif., USA); the monoclonal antibody toTsg101 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,Calif., USA); the monoclonal antibody to GFP was from ZYMED LaboratoriesInc. (South San Francisco, Calif., USA); the rabbit polyclonalantibodies Ab32 specific to α-adaptins and Ab20 to CALM were describedpreviously Sorkin et al (1995) Stoichiometric Interaction of theEpidermal Growth Factor Receptor with the Clathrin-associated ProteinComplex AP-2, J. Biol. Chem. 270:619, the polyclonal antibodies toclathrin light chains A and B were kindly provided by Dr. F. Brodsky(UCSF); monoclonal antibodies to PP1 (BD Transduction Laboratories) andα-Actinin (Chemicon) were kindly provided by Dr. M. Dell'Acqua(University of Colorado); Eps15 Ab577 and Eps15R Ab860 were kindlyprovided by Dr. P. P. Di Fiore (European Cancer Institute).

FIG. 11 demonstrates the in vivo functionality of 48 individual siRNAs,selected using Formula VIII (most of them will meet the criteriaincorporated by Formula IX as well) targeting 12 genes. Various celllines were transfected with siRNA duplexes (Dup1-4) or pools of siRNAduplexes (Pool), and the cells were lysed 3 days after transfection withthe exception of CALM (2 days) and β2 (4 days).

Note a β1-adaptin band (part of AP-1 Golgi adaptor complex) that runsslightly slower than β2 adaptin. CALM has two splice variants, 66 and 72kD. The full-length Eps15R (a doublet of ˜130 kD) and several truncatedspliced forms of ˜100 kD and ˜70 kD were detected in Eps15Rimmunoprecipitates (shown by arrows). The cells were lysed 3 days aftertransfection. Equal amounts of lysates were resolved by electrophoresisand blotted with the antibody specific to a targeted protein (GFPantibody for YFP fusion proteins) and the antibody specific to unrelatedproteins PP1 phosphatase or α-actinin, and TSG101. The amount of proteinin each specific band was normalized to the amount of non-specificproteins in each lane of the gel. Nearly all of them appear to befunctional, which establishes that Formula VIII and IX can be used topredict siRNAs' functionality in general in a genome wide manner.

To generate the fusion of yellow fluorescent protein (YFP) with Rab5b orRab5c (YFP-Rab5b or YFP-Rab5c), a DNA fragment encoding the full-lengthhuman Rab5b or Rab5c was obtained by PCR using Pfu polymerase(Stratagene) with a SacI restriction site introduced into the 5′ end anda KpnI site into the 3′ end and cloned into pEYFP-C1 vector (CLONTECH,Palo Alto, Calif., USA). GFP-CALM and YFP-Rab5a were describedpreviously Tebar, Bohlander, & Sorkin (1999) Clathrin Assembly LymphoidMyeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits,Interactions with Clathrin, and the Impact of Overexpression onClathrin-mediated Traffic, Mol. Biol. Cell 10:2687.

Example IV Validation of the Algorithm Using Eg5, GADPH, ATE1, MEK2,MEK1, QB, LaminA/C, c-myc, Human Cyclophilin, and Mouse Cyclophilin

A number of genes have been identified as playing potentially importantroles in disease etiology. Expression profiles of normal and diseasedkidneys has implicated Edg5 in immunoglobulin A neuropathy, a commonrenal glomerular disease. Myc1, MEK1/2 and other related kinases havebeen associated with one or more cancers, while lamins have beenimplicated in muscular dystrophy and other diseases. For these reasons,siRNA directed against the genes encoding these classes of moleculeswould be important research and therapeutic tools.

FIG. 12 illustrates four siRNAs targeting 10 different genes (Table Vfor sequence and accession number information) that were selectedaccording to the Formula VIII and assayed as individuals and pools inHEK293 cells. The level of siRNA induced silencing was measured usingthe B-DNA assay. These studies demonstrated that thirty-six out of theforty individual SMART-selected siRNA tested are functional (90%) andall 10 pools are fully functional.

Example V Validation of the Algorithm Using Bcl2

Bcl-2 is a ˜25 kD, 205-239 amino acid, anti-apoptotic protein thatcontains considerable homology with other members of the BCL familyincluding BCLX, MCL1, BAX, BAD, and BIK. The protein exists in at leasttwo forms (Bcl2a, which has a hydrophobic tail for membrane anchorage,and Bcl2b, which lacks the hydrophobic tail) and is predominantlylocalized to the mitochondrial membrane. While Bcl2 expression is widelydistributed, particular interest has focused on the expression of thismolecule in B and T cells. Bcl2 expression is down-regulated in normalgerminal center B cells yet in a high percentage of follicularlymphomas, Bcl2 expression has been observed to be elevated. Cytologicalstudies have identified a common translocation ((14;18)(q32;q32))amongst a high percentage (>70%) of these lymphomas. This genetic lesionplaces the Bcl2 gene in juxtaposition to immunoglobulin heavy chain gene(IgH) encoding sequences and is believed to enforce inappropriate levelsof gene expression, and resistance to programmed cell death in thefollicle center B cells. In other cases, hypomethylation of the Bcl2promoter leads to enhanced expression and again, inhibition ofapoptosis. In addition to cancer, dysregulated expression of Bcl-2 hasbeen correlated with multiple sclerosis and various neurologicaldiseases.

The correlation between Bcl-2 translocation and cancer makes this genean attractive target for RNAi. Identification of siRNA directed againstthe bcl2 transcript (or Bcl2-IgH fusions) would further ourunderstanding Bcl2 gene function and possibly provide a futuretherapeutic agent to battle diseases that result from altered expressionor function of this gene.

In Silico Identification of Functional siRNA

To identify functional and hyperfunctional siRNA against the Bcl2 gene,the sequence for Bcl-2 was downloaded from the NCBI UNIGENE database andanalyzed using the Formula VIII algorithm. As a result of theseprocedures, both the sequence and-SMARTSCORES™, or siRNA rankings, ofthe Bcl2 siRNA were obtained and ranked according to theirfunctionality. Subsequently, these sequences were BLAST'ed (database) toinsure that the selected sequences were specific and contained minimaloverlap with unrelated genes. The SMARTSCORES™, or siRNA rankings, forthe top 10 Bcl-2 siRNA are identified in FIG. 13.

In Vivo Testing of Bcl-2 siRNA

Bcl-2 siRNAs having the top ten SMARTSCORES™, or siRNA rankings, wereselected and tested in a functional assay to determine silencingefficiency. To accomplish this, each of the ten duplexes weresynthesized using 2′-O-ACE chemistry and transfected at 100 nMconcentrations into cells. Twenty-four hours later assays were performedon cell extracts to assess the degree of target silencing. Controls usedin these experiments included mock transfected cells, and cells thatwere transfected with a non-specific siRNA duplex.

The results of these experiments are presented below (and in FIG. 14)and show that all ten of the selected siRNA induce 80% or bettersilencing of the Bcl2 message at 100 nM concentrations. These dataverify that the algorithm successfully identified functional Bcl2 siRNAand-provide a set of functional agents that can be used in experimentaland therapeutic environments.

siRNA 1 GGGAGAUAGUGAUGAAGUA SEQ ID NO 302 siRNA 2 GAAGUACAUCCAUUAUAAGSEQ ID NO 303 siRNA 3 GUACGACAACCGGGAGAUA SEQ ID NO 304 siRNA 4AGAUAGUGAUGAAGUACAU SEQ ID NO 305 siRNA 5 UGAAGACUCUGCUCAGUUUSEQ ID NO 306 siRNA 6 GCAUGCGGCCUCUGUUUGA SEQ ID NO 307 siRNA 7UGCGGCCUCUGUUUGAUUU SEQ ID NO 308 siRNA 8 GAGAUAGUGAUGAAGUACASEQ ID NO 309 siRNA 9 GGAGAUAGUGAUGAAGUAC SEQ ID NO 310 siRNA 10GAAGACUCUGCUCAGUUUG SEQ ID NO 311

Bcl2 siRNA: Sense Strand, 5′ to 3′

Example VI Sequences Selected by the Algorithm

Sequences of the siRNAs selected using Formulas (Algorithms) VIII and IXwith their corresponding ranking, which have been evaluated for thesilencing activity in vivo in the present study (Formula VIII and IX,respectively).

TABLE V ACCESSION FORMULA FORMULA GENE NAME NUMBER SEQ ID NOFTLLSEQTENCE VIII IX CLTC NM_004859 312 GAAAGAAUCUGUAGAGAAA 76 94.2 CLTCNM_004859 313 GCAAUGAGCUGUUUGAAGA 65 39.9 CLTC NM_004859 314UGACAAAGGUGGAUAAAUU 57 38.2 CLTC NM_004859 315 GGAAAUGGAUCUCUUUGAA 5449.4 CLTA NM_001833 316 GGAAAGUAAUGGUCCAACA 22 55.5 CLTA NM_001833 317AGACAGUUAUGCAGCUAUU 4 22.9 CLTA NM_001833 318 CCAAUUCUCGGAAGCAAGA 1 17CLTA NM_001833 319 GAAAGUAAUGGUCCAACAG −1 −13 CLTB NM_001834 320GCGCCAGAGUGAACAAGUA 17 57.5 CLTB NM_001834 321 GAAGGUGGCCCAGCUAUGU 15−8.6 CLTB NM_001834 322 GGAACCAGCGCCAGAGUGA 13 40.5 CLTB NM_001834 323GAGCGAGAUUGCAGGCAUA 20 61.7 CALM U45976 324 GUUAGUAUCUGAUGACUUG 36 −34.6CALM U45976 325 GAAAUGGAACCACUAAGAA 33 46.1 CALM U45976 326GGAAAUGGAACCACUAAGA 30 61.2 CALM U45976 327 CAACUACACUUUCCAAUGC 28 6.8EPS15 NM_001981 328 CCACCAAGAUUUCAUGAUA 48 25.2 EPS15 NM_001981 329GAUCGGAACUCCAACAAGA 43 49.3 EPS15 NM_001981 330 AAACGGAGCUACAGAUUAU 3911.5 EPS15 NM_001981 331 CCACACAGCAUUCUUGUAA 33 −23.6 EPS15R NM_021235332 GAAGUUACCUUGAGCAAUC 48 33 EPS15R NM_021235 333 GGACUUGGCCGAUCCAGAA27 33 EPS15R NM_021235 334 GCACUUGGAUCGAGAUGAG 20 1.3 EPS15R NM_021235335 CAAAGACCAAUUCGCGUUA 17 27.7 DNM2 NM_004945 336 CCGAAUCAAUCGCAUCUUC 6−29.6 DNM2 NM_004945 337 GACAUGAUCCUGCAGUUCA 5 −14 DNM2 NM_004945 338GAGCGAAUCGUCACCACUU 5 24 DNM2 NM_004945 339 CCUCCGAGCUGGCGUCUAC −4 −63.6ARF6 AF93885 340 UCACAUGGUUAACCUCUAA 27 −21.1 ARF6 AF93885 341GAUGAGGGACGCCAUAAUC 7 −38.4 ARF6 AF93885 342 CCUCUAACUACAAAUCUUA 4 16.9ARF6 AF93885 343 GGAAGGUGCUAUCCAAAAU 4 11.5 RAB5A BC001267 344GCAAGCAAGUCCUAACAUU 40 25.1 RAB5A BC001267 345 GGAAGAGGAGUAGACCUUA 1750.1 RAB5A BC001267 346 AGGAAUCAGUGUUGUAGUA 16 11.5 RAB5A BC001267 347GAAGAGGAGUAGACCUUAC 12 7 RAB5B NM_002868 348 GAAAGUCAAGCCUGGUAUU 14 18.1RAB5B NM_002868 349 AAAGUCAAGCCUGGUAUUA 6 −17.8 RAB5B NM_002868 350GCUAUGAACGUGAAUGAUC 3 −21.1 RAB5B NM_002868 351 CAAGCCUGGUAUUACGUUU −7−37.5 RAB5C AF141304 352 GGAACAAGAUCUGUCAAUU 38 51.9 RAB5C AF141304 353GCAAUGAACGUGAACGAAA 29 43.7 RAB5C AF141304 354 CAAUGAACGUGAACGAAAU 1843.3 RAB5C AF141304 355 GGACAGGAGCGGUAUCACA 6 18.2 EEA1 XM_018197 356AGACAGAGCUUGAGAAUAA 67 64.1 EEA1 XM_018197 357 GAGAAGAUCUUUAUGCAAA 6048.7 EEA1 XM_018197 358 GAAGAGAAAUCAGCAGAUA 58 45.7 EEA1 XM_018197 359GCAAGUAACUCAACUAACA 56 72.3 AP2B1 NM_001282 360 GAGCUAAUCUGCCACAUUG 49−12.4 AP2B1 NM_001282 361 GCAGAUGAGUUACUAGAAA 44 48.9 AP2B1 NM_001282362 CAACUUAAUUGUCCAGAAA 41 28.2 AP2B1 NM_001282 363 CAACACAGGAUUCUGAUAA33 −5.8 PLK NM_005030 364 AGAUUGUGCCUAAGUCUCU −35 −3.4 PLK NM_005030 365AUGAAGAUCUGGAGGUGAA 0 −4.3 PLK NM_005030 366 UUUGAGACUUCUUGCCUAA −5−27.7 PLK NM_005030 367 AGAUCACCCUCCUUAAAUA 15 72.3 GAPDH NM_002046 368CAACGGAUUUGGUCGUAUU 27 −2.8 GAPDH NM_002046 369 GAAAUCCCAUCACCAUCUU 243.9 GAPDH NM_002046 370 GACCUCAACUACAUGGUUU 22 −22.9 GAPDH NM_002046 371UGGUUUACAUGUUCCAAUA 9 9.8 c-Myc 372 GAAGAAAUCGAUGUUGUUU 31 −11.7 c-Myc373 ACACAAACUUGAACAGCUA 22 51.3 c-Myc 374 GGAAGAAAUCGAUGUUGUU 18 26c-Myc 375 GAAACGACGAGAACAGUUG 18 −8.9 MAP2K1 NM_002755 376GCACAUGGAUGGAGGUUCU 26 16 MAP2K1 NM_002755 377 GCAGAGAGAGCAGAUUUGA 160.4 MAP2K1 NM_002755 378 GAGGUUCUCUGGAUCAAGU 14 15.5 MAP2K1 NM_002755379 GAGCAGAUUUGAAGCAACU 14 18.5 MAP2K2 NM_030662 380 CAAAGACGAUGACUUCGAA37 26.4 MAP2K2 NM_030662 381 GAUCAGCAUUUGCAUGGAA 24 −0.7 MAP2K2NM_030662 382 UCCAGGAGUUUGUCAAUAA 17 −4.5 MAP2K2 NM_030662 383GGAAGCUGAUCCACCUUGA 16 59.2 KNSL1(EG5) NM_004523 384 GCAGAAAUCUAAGGAUAUA53 35.8 KNSL1(EG5) NM_004523 385 CAACAAGGAUGAAGUCUAU 50 18.3 KNSL1(EG5)NM_004523 386 CAGCAGAAAUCUAAGGAUA 41 32.7 KNSL1(EG5) NM_004523 387CUAGAUGGCUUUCUCAGUA 39 3.9 CyclophilinA NM_021130 388AGACAAGGUCCCAAAGACA −16 58.1 CyclophilinA NM_021130 389GGAAUGGCAAGACCAGCAA −6 36 CyclophilinA NM_021130 390 AGAAUUAUUCCAGGGUUUA−3 16.1 CyclophilinA NM_021130 391 GCAGACAAGGUCCCAAAGA 8 8.9 LAMIN A/CNM_170707 392 AGAAGCAGCUUCAGGAUGA 31 38.8 LAMIN A/C NM_170707 393GAGCUUGACUUCCAGAAGA 33 22.4 LAMIN A/C NM_170707 394 CCACCGAAGUUCACCCUAA21 27.5 LAMIN A/C NM_170707 395 GAGAAGAGCUCCUCCAUCA 55 30.1 CyclophilinBM60857 396 GAAAGAGCAUCUACGGUGA 41 83.9 CyclophilinB M60857 397GAAAGGAUUUGGCUACAAA 53 59.1 CyclophilinB M60857 398 ACAGCAAAUUCCAUCGUGU−20 28.8 CyclophilinB M60857 399 GGAAAGACUGUUCCAAAAA 2 27 DBI1 NM_020548400 CAACACGCCUCAUCCUCUA 27 −7.6 DBI2 NM_020548 401 CAUGAAAGCUUACAUCAAC25 −30.8 DBI3 NM_020548 402 AAGAUGCCAUGAAAGCUUA 17 22 DBI4 NM_020548 403GCACAUACCGCCUGAGUCU 15 3.9 rLUC1 404 GAUCAAAUCUGAAGAAGGA 57 49.2 rLUC2405 GCCAAGAAGUUUCCUAAUA 50 13.7 rLUC3 406 CAGCAUAUCUUGAACCAUU 41 −2.2rLUC4 407 GAACAAAGGAAACGGAUGA 39 29.2 SeAP1 NM_031313 408CGGAAACGGUCCAGGCUAU 6 26.9 SeAP2 NM_031313 409 GCUUCGAGCAGACAUGAUA 4−11.2 SeAP3 NM_031313 410 CCUACACGGUCCUCCUAUA 4 4.9 SeAP4 NM_031313 411GCCAAGAACCUCAUCAUCU 1 −9.9 fLUC1 412 GAUAUGGGCUGAAUACAAA 54 40.4 fLUC2413 GCACUCUGAUUGACAAAUA 47 54.7 fLUC3 414 UGAAGUCUCUGAUUAAGUA 46 34.5fLUC4 415 UCAGAGAGAUCCUCAUAAA 40 11.4 mCyclo_1 NM_008907 416GCAAGAAGAUCACCAUUUC 52 46.4 mCyclo_2 NM_008907 417 GAGAGAAAUUUGAGGAUGA36 70.7 mCyclo_3 NM_008907 418 GAAAGGAUUUGGCUAUAAG 35 −1.5 mCyclo_4NM_008907 419 GAAAGAAGGCAUGAACAUU 27 10.3 BCL2_1 NM_000633 420GGGAGAUAGUGAUGAAGUA 21 72 BCL2_2 NM_000633 421 GAAGUACAUCCAUUAUAAG 1 3.3BCL2_3 NM_000633 422 GUACGACAACCGGGAGAUA 1 35.9 BCL2_4 NM_000633 423AGAUAGUGAUGAAGUACAU −12 22.1 BCL2_5 NM_000633 424 UGAAGACUCUGCUCAGUUU 3619.1 BCL2_6 NM_000633 425 GCAUGCGGCCUCUGUUUGA 5 -9.7 QB1 NM_003365.1 426GCACACAGCUUACUACAUC 52 −4.8 QB2 NM_003365.1 427 GAAAUGCCCUGGUAUCUCA 4922.1 QB3 NM_003365.1 428 GAAGGAACGUGAUGUGAUC 34 22.9 QB4 NM_003365.1 429GCACUACUCCUGUGUGUGA 28 20.4 ATE1-1 NM_007041 430 GAACCCAGCUGGAGAACUU 4515.5 ATE1-2 NM_007041 431 GAUAUACAGUGUGAUCUUA 40 12.2 ATE1-3 NM_007041432 GUACUACGAUCCUGAUUAU 37 32.9 ATE1-4 NM_007041 433 GUGCCGACCUUUACAAUUU35 18.2 EGFR-1 NM_005228 434 GAAGGAAACUGAAUUCAAA 68 79.4 EGFR-1NM_005228 435 GGAAAUAUGUACUACGAAA 49 49.5 EGFR-1 NM_005228 436CCACAAAGCAGUGAAUUUA 41 7.6 EGFR-1 NM_005228 437 GUAACAAGCUCACGCAGUU 4025.9

Example VII Genome-Wide Application of the Algorithm

The examples described above demonstrate that the algorithm(s) cansuccessfully identify functional siRNA and that these duplexes can beused to induce the desirable phenotype of transcriptional knockdown orknockout. Each gene or family of genes in each organism plays animportant role in maintaining physiological homeostasis and thealgorithm can be used to develop functional, highly functional, orhyperfunctional siRNA to each gene. To accomplish this for the humangenome, the entire online NCBI REFSEQ database was accessed throughENTREZ (EFETCH). The database was processed through Formula VIII. Foreach gene the top 80-100 scores for siRNAs were obtained and BLAST'ed toinsure that the selected sequences are specific in targeting the gene ofchoice. These sequences are provided on the compact disks in electronicform in parent application Ser. No. 10/714,333.

Many of the genes to which the described siRNA are directed playcritical roles in disease etiology. For this reason, the siRNA listed inthe compact disk may potentially act as therapeutic agents. A number ofprophetic examples follow and should be understood in view of the siRNAthat are identified on the compact disk. To isolate these siRNA, theappropriate message sequence for each gene is analyzed using one of thebefore mentioned formulas (preferably formula VIII) to identifypotential siRNA targets. Subsequently these targets are BLAST'ed toeliminate homology with potentially off-targets.

The siRNA sequences listed above are presented in a 5′→3′ sense stranddirection. In addition, siRNA directed against the targets listed aboveas well as those directed against other targets and listed in thecompact disk may be useful as therapeutic agents.

Example VIII Evidence for the Benefits of Pooling

Evidence for the benefits of pooling have been demonstrated using thereporter gene, luciferase. Ninety siRNA duplexes were synthesized usingDharmacon proprietary ACE® chemistry against one of the standardreporter genes: firefly luciferase. The duplexes were designed to starttwo base pairs apart and to cover approximately 180 base pairs of theluciferase gene (see sequences in Table IV). Subsequently, the siRNAduplexes were co-transfected with a luciferase expression reporterplasmid into HEK293 cells using standard transfection protocols andluciferase activity was assayed at 24 and 48 hours.

Transfection of individual siRNAs showed standard distribution ofinhibitory effect. Some duplexes were active, while others were not.FIG. 15 represents a typical screen of ninety siRNA duplexes (SEQ ID NO032-120) positioned two base pairs apart. As the figure suggests, thefunctionality of the siRNA duplex is determined more by a particularsequence of the oligonucleotide than by the relative oligonucleotideposition within a gene or excessively sensitive part of the mRNA, whichis important for traditional anti-sense technology.

When two continuous oligonucleotides were pooled together, a significantincrease in gene silencing activity was observed (see FIGS. 16A and16B). A gradual increase in efficacy and the frequency of poolsfunctionality was observed when the number of siRNAs increased to 3 and4 (see FIGS. 16A, 16B, 17A, and 17B). Further, the relative positioningof the oligonucleotides within a pool did not determine whether aparticular pool was functional (see FIGS. 18A and 18B, in which 100% ofpools of oligonucleotides distanced by 2, 10 and 20 base pairs werefunctional).

However, relative positioning may nonetheless have an impact. Anincreased functionality may exist when the siRNA are positionedcontinuously head to toe (5′ end of one directly adjacent to the 3′ endof the others).

Additionally, siRNA pools that were tested performed at least as well asthe best oligonucleotide in the pool, under the experimental conditionswhose results are depicted in FIG. 19. Moreover, when previouslyidentified non-functional and marginally (semi) functional siRNAduplexes were pooled together in groups of five at a time, a significantfunctional cooperative action was observed (see FIG. 20). In fact, poolsof semi-active oligonucleotides were 5 to 25 times more functional thanthe most potent oligonucleotide in the pool. Therefore, pooling severalsiRNA duplexes together does not interfere with the functionality of themost potent siRNAs within a pool, and pooling provides an unexpectedsignificant increase in overall functionality

Example IX Pooling Across Species

Experiments were performed on the following genes: β-galactosidase,Renilla luciferase, and Secreted alkaline phosphatase, whichdemonstrates the benefits of pooling (see FIG. 21). Approximately 50% ofindividual siRNAs designed to silence the above-specified genes werefunctional, while 100% of the pools that contain the same siRNA duplexeswere functional.

Example X Highly Functional siRNA

Pools of five siRNAs in which each two siRNAs overlap to 10-90% resultedin 98% functional entities (>80% silencing). Pools of siRNAs distributedthroughout the mRNA that were evenly spaced, covering an approximate20-2000 base pair range, were also functional. When the pools of siRNAwere positioned continuously head to tail relative to mRNA sequences andmimicked the natural products of Dicer cleaved long double stranded RNA,98% of the pools evidenced highly functional activity (>95% silencing).

Example XI Human Cyclophyline

Table IV above lists the siRNA sequences for the human cyclophylineprotein. A particularly functional siRNA may be selected by applyingthese sequences to any of Formula I to VII above.

Alternatively, one could pool 2, 3, 4, 5 or more of these sequences tocreate a kit for silencing a gene. Preferably, within the kit therewould be at least one sequence that has a relatively high predictedfunctionality when any of Formulas I-VII is applied.

Example XII Sample Pools of siRNAS and Their Application to HumanDisease

The genetic basis behind human disease is well documented and siRNA maybe used as both research or diagnostic tools and therapeutic agents,either individually or in pools. Genes involved in signal transduction,the immune response, apoptosis, DNA repair, cell cycle control, and avariety of other physiological functions have clinical relevance andtherapeutic agents that can modulate expression of these genes mayalleviate some or all of the associated symptoms. In some instances,these genes can be described as a member of a family or class of genesand siRNA (randomly, conventionally, or rationally designed) can bedirected against one or multiple members of the family to induce adesired result.

To identify rationally designed siRNA to each gene, the sequence wasanalyzed using Formula VIII to identify a SMARTpool containing thefunctional sequences. To confirm the activity of these sequences, thesiRNA are introduced into a cell type of choice (e.g. HeLa cells, HEK293cells) and the levels of the appropriate message are analyzed using oneof several art proven techniques. siRNA having heightened levels ofpotency can be identified by testing each of the before mentionedduplexes at increasingly limiting concentrations. Similarly, siRNAhaving increased levels of longevity can be identified by introducingeach duplex into cells and testing functionality at 24, 48, 72, 96, 120,144, 168, and 192 hours after transfection. Agents that induce >95%silencing at sub-nanomolar concentrations and/or induce functionallevels of silencing for >96 hours are considered hyperfunctional.

Example XIII Validation of Multigene Knocking Using Rab5 and Eps

Two or more genes having similar, overlapping functions often leads togenetic redundancy. Mutations that knockout only one of, e.g., a pair ofsuch genes (also referred to as homologs) results in little or nophenotype due to the fact that the remaining intact gene is capable offulfilling the role of the disrupted counterpart. To fully understandthe function of such genes in cellular physiology, it is often necessaryto knockout or knockdown both homologs simultaneously. Unfortunately,concomitant knockdown of two or more genes is frequently difficult toachieve in higher organisms (e.g. mice) thus it is necessary tointroduce new technologies dissect gene function. One such approach toknocking down multiple genes simultaneously is by using siRNA. Forexample, FIG. 11 showed that rationally designed siRNA directed againsta number of genes involved in the clathrin-mediated endocytosis pathwayresulted in significant levels of protein reduction (e.g. >80%). Todetermine the effects of gene knockdown on clathrin-related endocytosis,internalization assays were performed using epidermal growth factor andtransferrin. Specifically, mouse receptor-grade EGF (CollaborativeResearch Inc.) and iron-saturated human transferrin (Sigma) wereiodinated as described previously (Jiang, X., Huang, F., Marusyk, A. &Sorkin, A. (2003) Mol Biol Cell 14, 858-70). HeLa cells grown in 12-welldishes were incubated with ¹²⁵I-EGF (1 ng/ml) or ¹²⁵I-transferrin (1μg/ml) in binding medium (DMEM, 0.1% bovine serum albumin) at 37° C.,and the ratio of internalized and surface radioactivity was determinedduring 5-min time course to calculate specific internalization rateconstant k_(e) as described previously (Jiang, X et al.). Themeasurements of the uptakes of radiolabeled transferrin and EGF wereperformed using short time-course assays to avoid influence of therecycling on the uptake kinetics, and using low ligand concentration toavoid saturation of the clathrin-dependent pathway (for E G F Lund, K.A., Opresko, L. K., Strarbuck, C., Walsh, B. J. & Wiley, H. S. (1990) J.Biol. Chem. 265, 15713-13723).

The effects of knocking down Rab5a, 5b, 5c, Eps, or Eps 15R(individually) are shown in FIG. 22 and demonstrate that disruption ofsingle genes has little or no effect on EGF or Tfn internalization. Incontrast, simultaneous knock down of Rab5a, 5b, and 5c, or Eps and Eps15R, leads to a distinct phenotype (note: total concentration of siRNAin these experiments remained constant with that in experiments in whicha single siRNA was introduced, see FIG. 23). These experimentsdemonstrate the effectiveness of using rationally designed siRNA toknockdown multiple genes and validates the utility of these reagents tooverride genetic redundancy.

Example XIV Validation of Multigene Targeting Using G6PD, GAPDH, PLK,and UQC

Further demonstration of the ability to knock down expression ofmultiple genes using rationally designed siRNA was performed using poolsof siRNA directed against four separate genes. To achieve this, siRNAwere transfected into cells (total siRNA concentration of 100 nM) andassayed twenty-four hours later by B-DNA. Results shown in FIG. 24 showthat pools of rationally designed molecules are capable ofsimultaneously silencing four different genes.

Example XV Validation of Multigene Knockouts as Demonstrated by GeneExpression Profiling, a Prophetic Example

To further demonstrate the ability to concomitantly knockdown theexpression of multiple gene targets, single siRNA or siRNA poolsdirected against a collection of genes (e.g. 4, 8, 16, or 23 differenttargets) are simultaneously transfected into cells and cultured fortwenty-four hours. Subsequently, mRNA is harvested from treated (anduntreated) cells and labeled with one of two fluorescent probes dyes(e.g. a red fluorescent probe for the treated cells, a green fluorescentprobe for the control cells.). Equivalent amounts of labeled RNA fromeach sample is then mixed together and hybridized to sequences that havebeen linked to a solid support (e.g. a slide, “DNA CHIP”). Followinghybridization, the slides are washed and analyzed to assess changes inthe levels of target genes induced by siRNA.

Example XVI Identifying Hyperfunctional siRNA

Identification of Hyperfunctional Bcl-2 siRNA

The ten rationally designed Bcl2 siRNA (identified in FIGS. 13, 14) weretested to identify hyperpotent reagents. To accomplish this, each of theten Bcl-2 siRNA were individually transfected into cells at a 300 pM(0.3 nM) concentrations. Twenty-four hours later, transcript levels wereassessed by B-DNA assays and compared with relevant controls. As shownin FIG. 25, while the majority of Bcl-2 siRNA failed to inducefunctional levels of silencing at this concentration, siRNA 1 and 8induced >80% silencing, and siRNA 6 exhibited greater than 90% silencingat this subnanomolar concentration.

Example XVII Gene Silencing: Prophetic Example

Below is an example of how one might transfect a cell.

Select a cell line. The selection of a cell line is usually determinedby the desired application. The most important feature to RNAi is thelevel of expression of the gene of interest. It is highly recommended touse cell lines for which siRNA transfection conditions have beenspecified and validated.

Plate the cells. Approximately 24 hours prior to transfection, plate thecells at the appropriate density so that they will be approximately70-90% confluent, or approximately 1×10⁵ cells/ml at the time oftransfection. Cell densities that are too low may lead to toxicity dueto excess exposure and uptake of transfection reagent-siRNA complexes.Cell densities that are too high may lead to low transfectionefficiencies and little or no silencing. Incubate the cells overnight.Standard incubation conditions for mammalian cells are 37° C. in 5% CO₂.Other cell types, such as insect cells, require different temperaturesand CO₂ concentrations that are readily ascertainable by persons skilledin the art. Use conditions appropriate for the cell type of interest.

siRNA re-suspension. Add 20 μl siRNA universal buffer to each siRNA togenerate a final concentration of 50 μM.

siRNA-lipid complex formation. Use RNase-free solutions and tubes. Usingthe following table, Table VI

TABLE VI 96-WELL 24-WELL MIXTURE 1 (TRANSIT-TKO-PLASMID DILUTIONMIXTURE) Opti-MEM 9.3 μl 46.5 μl TransIT-TKO (1 μg/μl) 0.5 μl 2.5 μlMIXTURE 1 10.0 μl 50.0 μl FINAL VOLUME MIXTURE 2 (SIRNA DILUTIONMIXTURE) Opti-MEM 9.0 μl 45.0 μl siRNA (1 μM) 1.0 μl 5.0 μl MIXTURE 210.0 μl 50.0 μl FINAL VOLUME MIXTURE 3 (SIRNA-TRANSFECTION REAGENTMIXTURE) Mixture 1 10 μl 50 μl Mixture 2 10 μl 50 μl MIXTURE 3 20 μl 100μl FINAL VOLUME Incubate 20 minutes at room temperature MIXTURE 4(MEDIA-SIRNA/TRANSFECTION REAGENT MIXTURE) Mixture 3 20 μl 100 μlComplete media 80 μl 400 μl MIXTURE 4 100 μl 500 μl FINAL VOLUMEIncubate 48 hours at 37° C.

Transfection. Create a Mixture 1 by combining the specified amounts ofOPTI-MEM serum free media and transfection reagent in a sterilepolystyrene tube. Create a Mixture 2 by combining specified amounts ofeach siRNA with OPTI-MEM media in sterile 1 ml tubes. Create a Mixture 3by combining specified amounts of Mixture 1 and Mixture 2. Mix gently(do not vortex) and incubate at room temperature for 20 minutes. Createa Mixture 4 by combining specified amounts of Mixture 3 to completemedia. Add appropriate volume to each cell culture well. Incubate cellswith transfection reagent mixture for 24-72 hours at 37° C. Thisincubation time is flexible. The ratio of silencing will remainconsistent at any point in the time period. Assay for gene silencingusing an appropriate detection method such as RT-PCR, Western blotanalysis, immunohistochemistry, phenotypic analysis, mass spectrometry,fluorescence, radioactive decay, or any other method that is now knownor that comes to be known to persons skilled in the art and that fromreading this disclosure would useful with the present invention. Theoptimal window for observing a knockdown phenotype is related to themRNA turnover of the gene of interest, although 24-72 hours is standard.Final Volume reflects amount needed in each well for the desired cellculture format. When adjusting volumes for a Stock Mix, an additional10% should be used to accommodate variability in pipetting, etc.Duplicate or triplicate assays should be carried out when possible.

Example XVIII siRNA Directed Against KRAS

The entire online NCBI REFSEQ database was accessed through ENTREZ(EFETCH). The database was processed through Formula VIII. For each genethe top 80-100 scores for siRNAs were obtained and BLAST'ed to insurethat the selected sequences are specific in targeting the gene ofchoice. The selected sequences so obtained included the following 26siRNAs directed against KRAS (v-Ki-ras2 Kirsten rat sarcoma viraloncogene homolog):

GGCAUCAUGUCCUAUAGUU, (SEQ ID NO 438) CGAAUAUGAUCCAACAAUA,(SEQ ID NO 439) GGACGAAUAUGAUCCAACA, (SEQ ID NO 440)UAAGGACUCUGAAGAUGUA, (SEQ ID NO 441) GGAAGCAAGUAGUAAUUGA,(SEQ ID NO 442) GCAAGAAGUUAUGGAAUUC, (SEQ ID NO 443)GAUGAUGCCUUCUAUACAU, (SEQ ID NO 444) GACAAAGUGUGUAAUUAUG,(SEQ ID NO 445) GAAACUGAAUACCUAAGAU, (SEQ ID NO 446)GCUCAGGACUUAGCAAGAA, (SEQ ID NO 447) AAACCUGUCUCUUGGAUAU,(SEQ ID NO 448) GAACUAGCAAUGCCUGUGA, (SEQ ID NO 449)GGAGGGCUUUCUUUGUGUA, (SEQ ID NO 450) GGAGAGAGGCCUGCUGAAA,(SEQ ID NO 451) GGGCUUUCUUUGUGUAUUU, (SEQ ID NO 452)GUUGAGACCUUCUAAUUGG, (SEQ ID NO 453) GCAUGCAGUUGAUUACUUC,(SEQ ID NO 454) CAAGACAGGGUGUUGAUGA, (SEQ ID NO 455)GAAUAUGAUCCAACAAUAG, (SEQ ID NO 456) UCAAAGACAAAGUGUGUAA,(SEQ ID NO 457) GUUAAGGACUCUGAAGAUG, (SEQ ID NO 458)UUUAUGGGCUUCCUGAUGA, (SEQ ID NO 459) CAUCAUGUCCUAUAGUUUG,(SEQ ID NO 460) UGCAGUUGAUUACUUCUUA, (SEQ ID NO 461)CGAUACAGCUAAUUCAGAA, (SEQ ID NO 462) and GAAGUUAUGGAAUUCCUUU(SEQ ID NO 463)

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departure from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1. An siRNA molecule, wherein said siRNA molecule consists of: (a) aduplex region; and (b) either no overhang regions or at least oneoverhang region, wherein each overhang region contains six or fewernucleotides, wherein the duplex region consists of a sense region and anantisense region, wherein said sense region and said antisense regiontogether form said duplex region and said duplex region is 19-30 basepairs in length and said antisense region comprises a sequence that isthe complement of a sequence selected from SEQ ID NOs: 438-447, 449-453,455-460 and
 463. 2. The siRNA molecule of claim 1, wherein saidantisense region and said sense region are each 19-25 bases in length.3. The siRNA molecule of claim 2, wherein said antisense region and saidsense region are each 19 bases in length.
 4. The siRNA molecule of claim1, wherein said siRNA molecule has at least one overhang region.
 5. ThesiRNA molecule of claim 1, wherein said siRNA molecule has no overhangregions.
 6. The siRNA molecule of claim 2, wherein said siRNA moleculehas at least one overhang region.
 7. The siRNA molecule of claim 2,wherein said siRNA molecule has no overhang regions.
 8. The siRNAmolecule of claim 3, wherein said siRNA molecule has at least oneoverhang region.
 9. The siRNA molecule of claim 3, wherein said siRNAmolecule has no overhang regions.
 10. A chemically synthesized doublestranded siRNA molecule, wherein: (a) each strand of said doublestranded siRNA molecule is between 19 and 30 nucleotides in length; and(b) one strand of said siRNA molecule comprises a sequence that is thecomplement of a sequence selected from SEQ ID NOs: 438-447, 449-453,455-460 and
 463. 11. The chemically synthesized double stranded siRNAmolecule of claim 10 wherein each strand of said siRNA molecule is 19-25nucleotides in length.
 12. The chemically synthesized double strandedsiRNA molecule of claim 11, wherein each strand of said siRNA moleculeis 19 nucleotides in length.
 13. A pool of at least two siRNAs, whereinsaid pool comprises a first siRNA and a second siRNA, wherein said firstsiRNA is the siRNA molecule of claim 1, wherein the duplex region ofsaid first siRNA is a first duplex region and said second siRNA consistsof a second duplex region and either no overhang regions or at least oneoverhang region, wherein each overhang region contains six or fewernucleotides, wherein the second duplex region of said second siRNAcomprises a sense region and an antisense region, wherein said senseregion and said antisense region of said second siRNA together form saidsecond duplex region and said second duplex region is 19-30 base pairsin length and said antisense region of said second duplex regioncomprises a sequence that is the complement of a sequence selected fromthe group consisting of a sequence selected from SEQ ID NOs: 438-447,449-453, 455-460 and
 463. 14. The pool of claim 13, wherein said firstsiRNA and said second siRNA each have no overhang regions.
 15. The poolof claim 13, wherein said first duplex region and said second duplexregion are each 19-25 base pairs in length.
 16. The pool of claim 15,wherein said first duplex region and said second duplex region are each19 base pairs in length.
 17. The pool of claim 16, wherein said firstsiRNA contains no overhang regions.
 18. The pool of claim 16, whereinthe second siRNA contains no overhang regions.
 19. The pool of claim 16,wherein the first siRNA contains at least one overhang region.
 20. Thepool of claim 16, wherein the second siRNA contains at least oneoverhang region.